Mass Spectrometry Lecture Slides

Chem 425- Mass Spectrometry
Tuesday and Thursday: 10am – 11:20am, C2-361

Richard W. Smith, Ph.D.
rw1smith@uwaterloo.ca
C2-264 (office) / C2-267 (lab)
UW Mass Spectrometry Facility
Course TA: Jonathan Martens

j2martens@uwaterloo.ca
Office Hour: Friday, 12:30pm-1:30pm, C2-269B
1
Course Overview
• 1. Introduction
• 2. History
• 3. Electron Ionization
– The mass spectrometer and mass calibration
– Electron Ionization (EI)
• Fragmentation
• QET
• Elemental composition, isotopic species, mass resolution, accurate mass, z>1,
rings plus double bonds, nitrogen rule
• Interpretation of EI mass spectra
• 4. Chemical Ionization (CI and DCI)

– Ion formation
– Thermochemical considerations and reagent systems
– Negative ion formation
2
Course Overview cont
• 5. Field Ionization and Field Desorption (FI and FD)
• 6. Particle Bombardment
– FAB, LSIMS and 252Cf
• 7. Laser Desorption Ionization and Matrix Assisted Laser Desorption

Ionization (LDI and MALDI)
• 8. Inductively Coupled Plasma (ICP)
• 9. Atmospheric Pressure Ionization (API)

– Electrospray (ESI)
– Atmospheric Pressure Chemical Ionization (APCI)
– Atmospheric Pressure Photo-Ionization (APPI)
3
• 10. Mass Separation
– Magnetic and electrostatic fields (B and E)
– Quadrupoles (Q and QQQ)
– 3D Quadrupole Ion Trap (QIT)
– 2D linear Ion Trap
– Time of Flight (Tof)
– Fourier Transform Ion Cyclotron Resonance (FTICR)
• 11. Hyphenation in Mass Spectrometry

– Gas Chromatography – Mass Spectrometry (GC-MS)
– Liquid Chromatography – Mass Spectrometry (LC-MS)
• Interfaces
– MS-MS and MSn - Tandem Mass Spectrometry
• Collision Induced Dissociation (CID)
• BE, QQQ, 3D QIT, 2D QIT, QTof……
4
• 12. Quantitation
– Primarily with QQQ in MRM/SRM mode
• 13. Ion detection and Focusing

– Faraday cup, Electron multiplier, multichannel plate and photomultiplier
• 14. Summary
5
• 70 minute mid-term exam during class (Thursday, February 28th) –
25%
• 150 minute final exam (to be scheduled) – 55%
• 10 Weekly Quizzes – total of 10%. Held at the end of class starting on
Tuesday, Jan 15th (~10min)
– 10 questions/quiz
– 7 to 10 correct answers 1 mark
– 3 to 6 correct answers 0.5 mark
– 0 to 2 correct answers 0 mark
• 12 minute seminar – 10%. Held weekly at the end of class starting on
Thursday, Jan 17
– Teams of 2 prepare and present the material
6
Course Calendar
January 2008
24 class sessions: Tuesday and Thursday – 10am to 11:20am in C2-361
Sun Mon Tues Wed Thurs Fri Sat
1 2 3 4 5
6 7 8 9 10 11 12
1. Introduction 3. EI
2. History
13 14 15 16 17 18 19
Quiz 1 Sem 1
3. EI 3. EI
20 21 22 23 24 25 26
Quiz 2 Sem 2
4. CI 4. CI
27 28 29 30 31
Quiz 3 Sem 3
5. FI and FD 6. Particle
Bombardment
7
Course Calendar
February 2008
1 2
3 4 5 6 7 8 9
Quiz 4 Sem 4
7. LDI & MALDI 8. ICP
10 11 12 13 14 15 16
Quiz 5 Sem 5
9. API 9. API
17 18 19 20 21 22 23
Reading Week
24 25 26 27 28 29
Quiz 6
9. API
Mid-Term
8
Course Calendar
March 2008

1
2 3 4 5 6 7 8
Sem 6 Quiz 7
10. Mass Sep 10. Mass Sep
9 10 11 12 13 14 15
Sem 7 Quiz 8
10. Mass Sep 11. Hyphenation
16 17 18 19 20 21 22
Sem 8 Quiz 9
11. Hyphenation 11. Hyphenation
23 24 25 26 27 28 29
Sem 9 Quiz 10
11. Hyphenation 12. Quantitation
30 31
9
Course Calendar
April 2008
1 2 3 4 5
Sem 10 Sem 11 &12
13. Ion Focusing 14. Summary
and Detection Q and A
6 7 8 9 10 11 12
Exams
Start
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30
10
Resources
Text Books
• Mass Spectrometry – A Textbook (on 1 hour hold at Davis Library)
Jurgen H. Gross - Springer 2004 – QD96M3G76
• Mass Spectrometry: A Foundation Course
K. Downard – Royal Society of Chemistry 2004 – QD96M3D69X2004
• Chemical Ionization Mass Spectrometry – 2nd edition
Alex G. Harrison – CRC Press 1992 – QD96M3H371992
• Interpretation of Mass Spectra
Fred W. McLafferty – University Science Books
Database website
• NIST Library of EI Mass Spectra:
http://webbook.nist.gov/chemistry/name-ser.html
11
Resources
Journals:
• Biomedical and Environmental Mass Spectrometry / Biological Mass
Spectrometry
• International Journal of Mass Spectrometry
• Journal of the American Society For Mass Spectrometry
• Mass Spectrometry Reviews
• Journal of Mass Spectrometry
• Rapid Communications in Mass Spectrometry
12
History
• 1913: J. J. Thomson – parabola spectrograph
– Detection of the neon isotopes 20 and 22
– Parallel electric and magnetic field
– Detection on photoplate
Gas +ve and -ve ions

Discharge recorded
ion source simultaneously!
Focusing
Mass separation using

magnetic and electric fields
1
Photoplate detector
J. J. Thomson: First Mass Spectrum of 20Ne
and 22Ne
Modern measurements of 20Ne:22Ne ~ 10:1
2
History
• 1918 – 1920: Dempster and Aston (Nobel prize in 1922)
– Focussing of ions
– Higher resolution (≈ 600)
– tandem electric and magnetic field
– Allowed the detection of 21Ne (0.3% abundance)
3
History
• Late teens to early 30‘s, MS primarily used for isotopic analyis of the stable
elements
• 1934: J. Mattauch and R. Herzog (Nobel prize)

– Double focusing instruments > resolution 6500
• 1936: Secondary Ion Mass Spectrometry introduced
• 1940: Westinghouse Electric begins development of a portable MS for

commercial sale
• 1942: E.O. Lawrence develops the “Calutron“ prep scale MS for separation of
uranium isoptopes – 235U
• 1943: CEC installs 1st commercial MS at the Atlantic Refining Company
• 1945: CEC introduces 1st analogue computer for data analysis

4
History
• 1946 -1947: 1st description of time-of-flight MS, Metropolitan Vickers and
General Electric begins manufacturing MS
• 1948: ICR MS “Omegatron“ is developed
• 1952: 1st A/D converter for data aquisition (CEC)
• 1953: W. Paul → Quadrupole mass spectrometer and ion trap detectors

– Nobel prize in 1989.
• 1954: 1st high resolution MS developed at Imperial Chemical Industries

(ICI)
• 1956: PA determinations made by MS, McClafferty rearrangment

described, steroids 1st analyzed by MS
5
History
• 1957: GC/MS 1st demonstrated at Phillip Morris
• 1958: CEC introduces the 1st digitizer. Bendix TOFMS introduced
• 1959: peptides and oligonucleotides sequeunced at MIT
• 1960: Bendix introduces 1st direct insertion probe

– CEC introduces 1st high res double focusing MS
• 1966: Tandem MS for ion-molecule studies is developed

– Chemical Ionization is developed at ESSO (Munson and Field)
• 1968: Electrospray Ionization introduced at Northwestern U for studying

macromolecules. Computer library searching used for identification of
6
unknown compounds
History
• 1969: 1st GC/MS with integrated computer introduced
• 1971: relectron time-of-flight develpoed in Lenningrad
• 1974: M.B. Comisarow, A.C. Marshall – FT-MS. APCI interface is developed for
LCMS
• 1975: mass spectrometers are placed on the Viking Mars missions
• 1979: Ion evaporation model developed (ESI)
• 1980: ICPMS is developed at Iowa State U. 1st commercial QQQs introduced.
• 1981: FAB introduced.
• 1983: A.L. Yergey, M.L. Vestal – Thermospray interface for LCMS announced.
7
History
• 1984: Yamashita and Fenn – Electrospray (Nobel prize, 2002). 1st
commercial ion trap is introduced. Particle Beam interface developed at
GIT.
• 1986: LC interfaced to MS with pneumaticaly assisted ESI.
• 1988: Karas and Hillenkamp – MALDI
• 1994: Micro- and nano- ESI are introduced
• In the past 10 years a wide variety of hybrid instruments have been

introduced along with great improvements in instrument capabilities and
sensitivity (attamole)
8
The Mass Spectrometric Process
*Field free region
with ion focusing
Sample Introduction Ion source * Mass Analyzer(s) * Detector
Separates ions based Indirect

on their m/z value detection
Gas or condensed Some form of Ions

phase sample + ionization
Integrated with some degree of computer control 1

The Mass Spectrometer
• Sample Introduction
• Batch - direct insertion probe, desorption probe,
heated inlet, infusion
• Chromatographic – GC or LC (SFC)
• All Ion sources produce either
• Odd electron ions eg M+. (EI, charge exchange CI)

• Even electron ions eg [M+H]+, [M-H]- and/or adducts
[M+Na]+
(CI, FAB/SIMS, ESI, APCI, APPI)
• Stability of these ions determines the observed mass
spectrum
2
The Mass Spectrometer
• All Mass Analyzers
• Separate ions according to their mass/charge ratio (m/z)
• This separation is accomplished in a variety of fashions eg by
momentum (BE/EB), time of flight (Tof), selective transmission
employing Rf and DC voltages (Q and IT), frequency of motion
(FTICR)
• MS/MS and MSn
• Continuous beam (Q, BE/EB) and pulsed beam (IT, Tof, FTICR)
• All Detectors
• Conversion of +ve and –ve ions to 2o electrons or photons
• Indirect detection
• Computer employed for instrument control, data acquisition

and processing 3
JEOL HX110 Double Focusing Mass Spectrometer (EB)
ESA
Magnet
Tuning
Console
Detector
Source and
Sample Introduction
4
Benchtop Quadrupole GC/MS system
MS GC
Courtesy of Agilent 5
Ion Types
• Molecular ions: [M]+•, [M]-• are ions that arrive intact at the
detector
– Stable – k < 105s-1
• Fragment ions: [F]+ formed in the source by direct bond

cleavage or rearrangement
– Unstable k > 106s-1
• Metastable ions, m* are ions that fragment outside the ion

source but before they arrive at the detector
– 105s-1 < k < 106s-1
6
Ion Types
Fragment ions
M+.
Metastable ion
M+. and fragment ions~0.3Da wide

Metastable ion~3Da wide!
7
Ion Types
• Odd electron (such as [M]+•) and even electron ions (such as
[M+H]+)
• Quasi-molecular ions, e.g. [M+H]+, [M-H]-, [M+NH4]+,
[M+Na]+, [M+Cl]- ……..
• Cluster ions, [2M+H]+, (proton bound dimer)
• Isobaric ions (same nominal mass)
• Isotopic species
8
Mass Calibration
• All mass spectrometers make ions, pass them through a region where they
are separated according to their m/z ratio and finally detected
• A mass calibration must be performed in order to convert arrival times of
ions at the detector into a m/z value
• This is achieved by introducing a reference compound(s) into the source
who’s masses are known and then comparing this uncalibrated mass
spectrum with a reference spectrum of the same compound(s). The
patterns are matched and the data system then can, in real time, convert
arrival times into m/z values.
• Reference compounds are many and varied eg PFK, PEG, CsI and other
clusters such as phosphoric acid. The compound(s) chosen will depend
upon the ionization mode employed.
9
Mass Calibration
• Perfluorkerosene (PFK) with the JEOL HX110
62.3 69.0
344.6 380.94
493.1 542.9
Software algorithm
Uncalibrated Calibrated
Uncalibrated ΔM at m/z 380.97 = 36.3Da Calibrated ΔM at m/z 380.97 = 0.03Da
10
Electron Ionization – The Source
Filament S
Source block N
Focus plates
Sample
e-
in
Repeller
Ion beam out -
M+. and fragment
Trap ions
S
N
magnet 11
Electron Ionization
• Source operates at high vacuum (10-6 mbar) and high temp
(~200oC)
• Electrons are produced by heating a filament with a few amps
ie thermionic emission from a hot wire (rhenium or tungsten)
• They are focused onto the trap using a magnetic field and
voltage - usually 70V (can be changed) therefore e- are said to
have 70eV translational energy
• Trap: Collects the electrons on the far side of the source.
Usually the trap current is set and the electronic circuitry
adjusts the filament emission to maintain this value
• Repeller: +ve relative to the source, pushes the +ve ions
formed toward and out the exit slit where they are further
accelerated into the mass analyzer region 12
Electron Ionization
• Magnetic field causes e- to spiral

increasing the chance of an interaction
with the sample species – higher
sensitivity
• Sample is introduced and must be
volatilized before ionization can occur – ie
dilute gas phase unimolecular
processes (same for EI and CI) 13
EI of Methyl Stearate at 70eV
Mwt=298.5095
Monoisotopic mass=298.2872
M+.
14
Electron Ionization
• We need to understand the processes involved if we hope

to use MS as a structural elucidation tool!
• Ionization
• Isotopic distribution
• Accurate mass
• Fragmentation
15
Ionization
• one of the most common forms of ionization
AB + e- AB+. + e- + e-
1o 1o 2o (slow)
A + + B. etc
ΔHf (AB+.) = IP + ΔHf (AB)
IP is the ionization potential of AB ie the energy required to remove

one electron
• note that radical cations (M+.) are initially formed
16
Ionization
• the term electron “impact” should be avoided as the electron does not
impact the molecule
• 70 eV electrons have a deBroglie wavelength associated with them of

about 1.4Angstrom which is on the order of a bond length. The interaction of
the electron “wave” with the molecule causes a disturbance which causes
excitation or ionization of the molecule
• Removal of the e- from a molecule can be considered to occur at:

σ-bond < π-bond < free electron pair
17
Probability of ionization as a function of electron
energy
• Maximum ionization efficiency of organic ions at 20 – 50 eV

• every species has it‘s own curve depending on it‘s ionization cross section
• Excess energy ( ~ 1 – 8 eV) can lead to substantial fragmentation 18
Electron Energy
• some of the energy of the electron
goes into ionizing the molecule
(~7-12 eV). There is still more than
enough energy available to cause
extensive fragmentation of
molecules - typical bond strengths
are 1 – 4 eV.
• The electron energy chosen must
be higher than the ionization
potential (IP) of the compound
• the higher the electron energy, the
more energy there is available for
fragmentation and therefore a EI spectra of β-lactam using 70 and 15
higher degree of fragmentation is eV electrons. Note the intensity scale.
usually observed.
19
Ionization
• The ionization potential (IP), is σ/
IP Polarizability
Molecule A2
the minimum energy required to (eV) (10-24cm3)
remove an electron from a
He 0.38 24.59 0.21
species. The ionization cross
Ne 0.62 21.56 0.40
section (σ) is a measure of the
Ar 3.52 15.76 1.64
relative ease of ionization of a
Kr 5.29 14.00 2.48
molecule. In general, this is
Xe 7.31 12.13 4.04
proportional to the polarizability
CH4 4.30 12.61 2.59
of the molecule.
C2H6 8.35 11.52 4.47
• Polarizability is the relative
C3H8 11.1 10.94 6.30
tendency of the electron cloud
C6H14 22.3 10.13 11.9
of an atom to be distorted from
C6H6 16.9 9.24 10.3
its normal shape by the
C10H8 - 8.14 16.3
presence of a nearby external
electric field 20
Appearance Energy (AE)
• For sufficiently energetic electrons, ionization may be accompanied by
bond cleavage:
AB + e- → A+ + B + 2e-
• Most of the energy exchanged creates electronic excitation (along with

ionization). Almost no M+. ions will be in the vibrational ground state.
“Some” of these vibrationally excited ions may be above the dissociation
energy level
• The appearance energy (AE) of A+ from AB is defined as the minimum

electron energy at which A+ is formed from AB. AE measurements are very
useful for determining standard heats of formation however requires
specialized instrumentation.
AE = ΔHf (A+) + ΔHf (B) – ΔHf (AB) 21
• For example: the appearance energy of CH2OH2+ by dissociative
ionization of HOCH2CH2OH (ethylene glycol), also producing neutral
formaldehyde (CH2O) was found to be 11.42 eV (JACS 1982, 104,
2931). What is the heat of formation of CH2OH2+ given that the
heats of formation of ethylene glycol and formaldehyde are –387.6
and –108.7 kJ mol-1, respectively?
AE = 11.42eV
HOCH2CH2OH CH2OH2+ + CH2O
1eV=96.485 kJ mol-1
Therefore 11.42 eV = 1102 kJ mol-1
22
AE = ΔHf (CH2OH2+) + ΔHf (CH2O) – ΔHf (HOCH2CH2OH)
or
ΔHf (CH2OH2+) = AE - ΔHf (CH2O) + ΔHf (HOCH2CH2OH)
ΔHf (CH2OH2+) = (1102 + 108.7 – 387.6) kJmol-1

= 823 kJmol-1
In fact, CH2OH2+ has been determined to be approximately
20 kJ mol-1 lower in energy than its conventional isomer
CH3OH+ (ΔHf = 844 kJmol-1).
23
Fragmentation in EI
• Both simple bond cleavages as well as complex
rearrangements may take place after EI
ie. simple bond cleavage, t-butyl chloride:
C(CH3)3Cl +e- C(CH3)3+ +Cl. +2e- 24

Fragmentation in EI
ie complex rearrangement (McLafferty Rearrangement)
O
+ e- C3H6O+ + C2H4 + 2e-
2-pentanone
H distonic radical cation

O .+ CH2 OH+
+
CH2 C
.CH CH3
2
McLafferty Rearrangement: any fragmentation that can be described as a

transfer of a γ-hydrogen to a double bonded atom through a six-membered
transition state with β-cleavage. Occurs in saturated aldehydes, ketones and
carboxylic acids
Distonic Radical Cation: a radical cation in which the charge and radical sites are
separated 25
Fragmentation in EI
Stevenson’s Rule:
AB+ e- A+ + B. + 2e-
or A. + B+ + 2e-
For simple cleavage, the species of lower ionization energy will give
the more abundant ion. Therefore:
If IE(A) < IE(B) then IA+ > IB+
31
EI of methanol
32
29
CH3+
IP=9.84eV OH+
IP=13eV
26
EI of acetone, (CH3)2CO
CH3CO+ 43
IP=7eV
M+.
m/z 58
CH3+
IP=9.84eV
27
Quasi-Equilibrium Theory (QET)
• A theoretical approach to describe the unimolecular decomposition

of ions
• In most mass spectrometers, processes occur in the highly diluted
gas phase therefore bimolecular reactions are rare
• isolated ions are not in thermal equilibrium with their surroundings
and can only internally redistribute energy by dissociation or
isomerization (rearrangement)
• The rate constant (k) of a unimolecular reaction is strongly
influenced by the excess energy (Eex) of the reactants in the
transition state
• Removal of an electron can be considered to occur at a σ-bond, π-
bond or at a free electron pair
• For charge localization: free electron pair > π-bond > σ-bond and
28
this is reflected in IE’s
Vertical Transitions
Energy
M.+ A+ + B.
dissociation
energy M+. stable Ionization is very fast
therefore termed to be
a vertical ionization
IE process.
r 0 r1 29
Dissociation coordinate
Take Away Messages from QET
• EI occurs very fast ~ 10-16s and is a vertical process (Franck-

Condon principle)
• The probability of a particular vertical transition from the neutral to

a certain vibrational level is described by the Franck-Condon
factors.
• The larger r1 compared to r0, the more probable will be the

generation of ions excited even above their dissociation level
• Ionization tends to cause weakening of the bonding (bond

lengthening) in the ion compared to it’s precursor neutral
• The shape of the potential energy surface will also influence the
ions fate 30
Interpretation of EI spectra:
Elemental Composition
The elemental composition:
• The use of isotope peaks

• High resolution for accurate mass determinations
• Rings + Double Bonds
• double bond equivalents (DBE)
31
Some Common Isotopic Species
Element Type is sometimes termed X, X+1, X+2 etc

32
Isotopic Classification of the Elements
Some Definitions:
• Atomic number specifies the number of protons in the nucleus eg C is

element 6 6C
• Atoms with the same atomic number but with different number of
neutrons are termed isotopes eg 17Cl 35Cl and 37Cl (3:1)
• The mass number is the sum of protons and neutrons in an atom

can be confusing for example both 18Ar and 20Ca have a mass
number of 40
• Elements are classified as: A 19F, 31P, 127I
A+1 12C and 13C

A+2 35Cl and 37Cl
A-1 6Li and 7Li
polyisotopic Sn 10 isotopes
33
Isotopic Distributions
Calculation of the abundance ratios of isotopic peaks of molecules

containing two isotopes, where the isotope natural abundances are given
as a and b and n is the number of this species in the molecule:
(a + b)n = an +nan-1b + n(n-1)an-2b2/(2!) +

n(n-1)(n-2) an-3b3/(3!) + …. (binominal equation)
e.g. if n = 2:
(a+b)² = a² + 2ab + b²
For two Cl atoms where a = 100 (35Cl) and b = 32 (37Cl):

100² + 2•100•32 + 32² = 100 : 64 : 10
Likewise if n=3: a3 + 3a2b +3ab2 + b3

and if n=4: a4 + 4a3b + 6a2b2 + 4ab3 +b4 34
Common Isotopic Distributions
C1 C10 C100 S1 S2 S3
10 10 100 100 100 100
%
%
%
%
0 m
as 0 m
as 0 m
ass 0 m
ass 0 m
ass 0 m
ass
12 13 19 120 121 1199 1200 1201 1202 1203 1204 1205 31 32 33 34 35 63 64 65 66 95 96 97 98 99
100
Si1 100 Si2 100
Si3 100
F1 100 F10
% % % % %
0 mass
35
0 m
ass 0 mass 0 m
ass 0 m
ass
27 28 29 30 31 55 56 57 58 82 83 84 85 86 87 18 19 20 21 188 189 190 191
Common Isotopic Distributions
100
Cl1 100
Cl2 100 Cl3 100 Cl4
% % % %
0 ass
m 0 mass 0 mass 0 mass
34 35 36 37 38 68 69 70 71 72 73 74 75 103 104 105 106 107 108 109 110 111 112 139 140 141 142 143 144 145 146
Br1 Br2 Br3 Br4

100
100 100 100
%
% % %
0
78 79 80 81 82
mass
0
157 158 159 160 161 162 163
mass 0
235 236 237 238 239 240 241 242 243 244
mass 0
314 315 316 317 318 319 320 321 322 323 324
mass 36
Isotopic Distributions at “High” m/z
C200H320N50O50S10
100
100
@FWHM=1
FWHM=0.4 ~ 10,200 Resn
0 mass
4536 4538 4540 4542 4544 4546 4548 4550 4552 4554 4556 4558
0 mass
4536 4538 4540 4542 4544 4546 4548 4550 4552 4554 4556 4558
C192H290N40O40S5Cl5Br5
100
10vs16 amu
100
0
37
mass
4524 4526 4528 4530 4532 4534 4536 4538 4540 4542 4544 4546 4548
0 mass
4524 4526 4528 4530 4532 4534 4536 4538 4540 4542 4544 4546 4548
Mass Resolution
• Mass resolution: represents the ability to separate two
adjacent masses. It measures the "sharpness" of the MS
peak.
Resolution = M/ΔM
Normally reported at
10% or 50% valley
•Mass accuracy: indicates the accuracy of the mass information

provided by the mass spectrometer 38
Mass Resolution
1188.5654
100
1189.5654
1190.5654
FWHM = 0.1
%
1191.5654
1192.5654
0
1188.5654
100
1189.5654
1190.5654
FWHM = 0.2
%
1191.5654
1192.5654
0
1188.5654
100
FWHM = 0.5
1189.5654
1190.5654
%
1191.5654
1192.5654
0
1188.6514
100
FWHM = 1
%
FWHM = 2
1189.1201
100
%
0
1183 1184 1185 1186 1187 1188 1189 1190 1191 1192 1193 1194 1195 1196 1197 39 mass
1198
Nominal vs Monoistopic* Mass
Nominal mass Acc mass Av Mass % Rel Int
1H 1 1.00783 99.98
1.00798
2H 2 2.01410 0.01
12C 12 12.00000 98.9
12.01104
13C 13 13.00335 1.1
14N 14 14.00307 99.63
14.00676
15N 15 15.00010 0.37
16O 16 15.99491 99.76
17O 17 16.99913 15.99933 0.04
18O 18 17.99916 0.2
19F 19 18.99840 18.99840 100*
32S 32 31.97207 95.02
33S 33 32.97146 0.75
32.06439
34S 34 33.96787 4.21
36S 36 35.96708 0.02
35Cl 35 34.96885 75.77
35.45274 40
37Cl 37 36.96590 24.23
Nominal vs Monoistopic vs Average Mass
Take for example: C19H31N4O4Cl1,nominal mass = 414
Monoisotopic mass = 414.2034
100.0
80.0
60.0
Average mass = 414.9267
40.0
20.0
0.0
412.00 414.00 416.00 418.00 420.00 422.00
41
Nominal vs Monoistopic vs Average Mass
Nominal Mass Average Mass Monoisotopic

(A) (B) Mass (C)
C18H36O2 284 284.47724 284.27152
C17H32S1O1 284 284.50138 284.21737
A: different elemental composition but same nominal mass –

isobaric ions
B: calculated using C= 12.01104, H= 1.00798, O= 15.99933 and
S= 32.06439
C: calculated using 12C=12.0000, 1H= 1.00783, 16O=15.99491
and 32S= 31.97207
42
Accurate Mass Determinations
• High resolution enables isobaric species to be

differentiated based on their non-integral accurate mass.
• Consider the example below: all have a nominal mass of
28 and are therefore considered to be isobaric:
CO = 27.9949
N2 = 28.0061
} ΔM = 0.0112 M/ΔM = 2500
C2H4 = 28.0313
} ΔM = 0.0252 M/ΔM = 1110
These isobaric ions can be differentiated based on their

accurate mass if sufficient mass resolution is employed
43
• Most often employed to determine the elemental

composition of an ion, usually [M]+. or [M+H]+ ion
• Experimental error minimized by co-introducing a reference
compound into the source along with the unknown ie
Perfluorokerosene (CxFy)
• PFK used as it is mass deficient ie 12C=12.00000 and 19F=
18.99840
• Error normally expressed as ppm or millimass units
(1mmu=0.001amu)
Mass Accuracy (ppm) =

(true mass - observed mass )
× 10 6
true mass 44
Mass Deficiency and Mass Sufficiency
• The isotopic mass is very close but not equal to the nominal
mass of that isotope:
• Therefore the calculated exact mass of a molecule or of a
monoisotopic ion equals it’s monoisotopic mass
• 12C is the only exception because the unified atomic mass (u)
is defined as 1/12 of the mass of 12C, 1u=1.66055x10-27kg
and 12C is arbitrarily assigned as 12.0000000
• As a consequence of these non-integer masses almost no
combination of atoms will have the same calculated exact
mass whereas they might have the same nominal mass, that
is, they are isobaric
45
• Mass Deficiency – the exact mass of an isotope or molecule is lower

than it’s nominal mass
– eg nominal mass = 352, exact mass = 351.9897
• Mass Sufficiency – the exact mass of an isotope or molecule is

higher than it’s nominal mass
– eg nominal mass = 352, exact mass = 352.0347
– Only H, He, Li, Be, B and N are mass sufficient
• Atomic masses up to O are slightly higher than the nearest whole

number while O and above are slightly less than the nearest whole
number
46
Proton, Neutron and Electron Mass
• Mass of proton : 1.6726 x 10-27 kg
• Mass of neutron: 1.6749 x 10-27 kg
• Mass of electron: 0.00091x10-27 kg
• Relative:
proton : neutron : electron
1 : 1.00138 : 0.0005
Example 12C:
6 protons + 6 neutrons + 6 electrons (1s2.2s2.2p2)
should = 12.01128 but in fact is 12.0107
WHY?
47
Mass Defect
• The mass of an atom is less than the total mass of the constituent
protons, neutrons and electrons
• Mass defect, Δm = (Σmi) – matom and should be negative!
• This missing mass can be explained by Einstein's theory of mass-energy

equivalence, E = mc2
• The deviations from whole numbers represents the energy required to
bind the atomic nucleus together
• Also known as the Binding Energy, BE = Δmc2
48
• The larger the nucleus the more energy is required and the more
mass deficient an isotope is:
– 4He 4.0026
– 20Ne 19.99244
– 40Ar 39.96238 Relative to 12C = 12.00000
– 84Kr 83.91152
– 132Xe 131.90415
• If the unified atomic mass (u) had been based on 1H = 1.00000 and
not 12C/12 = 1.00000 (1H = 1.0078) then all isotopes would be mass
deficient ie this is completely arbitrary and not based on some
fundamental property of matter
49
LR EI of mw=308 (Resn~950)
PFK
PFK
Resn~9200 308.1085
C16H20O4S1
1ppm error or 0.3mmu
PFK
50
Charge State (z>1): EI of Pyrene (70eV)
doubly charged pyrene, M++ @ m/z 101
M+. 202
100
101.5
51
EI of Pyrene at low electron energy: ~20eV
M+. 202
1stIE~7.4eV
2nd IE~16.6eV
Note: no M++ and no fragmentation
52
Multiply Charged Ions: z>>1
A
100
Z=1
A compound of mw A of ions are 1amu apart
formula CxHy
%
(Mass resolution is constant )
mass
100
A/2
For example A=500.4 Z=2
ions are 0.5 amu apart
%
• z=1, m/z=500.4/1 = 500.4
• z=2, m/z=500.4/2 = 250.2

mass
• z=3, m/z=500.4/3 = 166.8 100

A/3 Z=3
• z=10, m/z= ?
ions are 0.333 amu apart
%
53
mass
Rings + Double Bonds
Based on valence rules the following expression can be derived:
imax
r+d = 1 +0.5 ΣNi(Vi-2)
i
Where Ni is the # of atoms and Vi is the valence of an element

Using 0.5x(Vi-2): For monovalent elements (H, F, Cl, Br, I) = -0.5
For divalent elements (O, S, Se) = 0 and so don’t contribute to DBE
For trivalent elements (N,P) = +0.5
For tetravalent elements (C, Si, Ge) = +1
r+d = 1 - 0.5Nmono + 0.5Ntri + Ntetra + 1.5Npenta + 2Nhexa + ……
Restriction to formulas of the general type, CxHyNzOn reduces the

expression to the commonly cited form:
r+d = x – 0.5y + 0.5z + 1 54

Rings + Double Bonds
• a whole number for any OE ion

• a non-integer for an EE ion
• Significance
– Informs about ion type (OE or EE) and basic structural info
(number of rings + double bonds)
– For EE ions, subtract 0.5 to get the right number of rings +
double bonds
– Sometimes called double bond equivalents (DBE)
55
Nitrogen rule
• If a compound contains an even number of nitrogen atoms
(0,2,4,6…), its molecular ion will be an even mass; and if it has
an odd number (1,3,5…), it will have an odd mass
• Why nitrogen?
– With the exception of N, all elements having an odd # of
valences also have an odd mass (H, P, F, Cl etc)
– all elements having an even # of valences have an even
mass (C, O, S, Si, etc)
– N has an odd # of valences and an even mass
• Value: places constraints on the numbers of nitrogens present
• It will be the inverse for EE ions, (M+H)+ 56
Interpretation of EI spectra: The molecular ion, M+.
• Molecular ion, M+•, most valuable information (mass, isotopic
distribution, elemental composition)
• Odd electron ions, e.g. [M]+. ions, dissociate and form even
electron fragment ions and neutral radicals (direct bond
cleavages) or odd electron fragment ions and neutral molecules
(rearrangement reactions).
• Often not stable, not detectable M+• → CI, ESI
• Requirements for an ion to be the molecular ion:

•Highest mass in spectrum
•Odd electron ion
•High mass ions near M+• muss be explained by logical neutral losses -
mass losses of 4 –14 and 21-25 highly unlikely
57
Interpretation of EI spectra:
The molecular ion, M+.
• Application of the Nitrogen rule:

– If a compound contains an even number of nitrogen
atoms (0, 2, 4..) its molecular ion will have an even
number.
– If a compound contains an odd number of nitrogen
atoms (1, 3, 5..) its molecular ion will have an odd
number.
• Relative abundance of molecular ion reflects structure

and stability
58
Ion Stability
1,3-dimethyladamantane – C12H20
Dodecane – C12H26
M+.
M +.
M+.
Perylene – C20H12
59
Guidelines for Understanding Ion Fragmentation
• We specify the nature of the precursor ion as either

.
– Odd electron ions (OE+ )
– Even electron ions (EE+)
• Favorability of ionization sites parallels bond stability

(lowest ionization energy, highest proton affinity)
– Sigma < pi, < nonbonding electrons (lone pairs)
• These ions can undergo the following processes, in

various combinations
– Radical-site initiation
– Charge-site initiation
– Rearrangements
– Charge retention/charge migration
60
Electron Ionization: basic mechanisms of ion
fragmentation
• Fragmentation in an EI source always unimolecular (low

pressure, no collision)
• Fragmentation stepwise (consecutive):

[ABCD]+• → [ABC]+ + D•
[AB]+ + C•
• Fragmentation by direct bond cleavage (leads to even electron ions):

[ABCD]+• → [AB]+ + CD•
example: CH3COCH3+• → CH3CO+ + CH3•
• or fragmentation by rearrangement (leads to odd electron ions):

[ABCD]+• → [AD]+• + BC
example: CH3COCH3+• → CH2=CO+. + CH4
61
Basic factors that influence the ion abundance
•Stability of product ions
•Stability of the neutral (neutral loss)

Neutral can be a neutral molecule or a neutral radical, where the
molecule is always more stable than the radical
In general the heat of formation of the ion is much higher than that of
the neutral (i.e. the ion is more important than the neutral)
•Stevensons rule (1951):

If their are two sets of ions and neutral radicals, such as
[ABCD]+• → [ABC]+ + D• or
[ABCD]+• → [ABC] • + D+
than the ion of lower ionization potential will be formed preferentially
•Loss of the largest alkyl radical:

If in an ion several different alkyl groups are bound to a carbon, the
largest alkyl group is lost preferentially (stability of the neutral is of
importance) 62
Basic Factors that Influence Ion Abundance:
Loss of Largest Radical
Example: 3-methyl-3-hexanol
CH3 CH3 CH3 C4H9

+.
C2H5 C C4H9 C2H5 C+ > C4H9 C+ > C2H5 C+
OH OH OH OH
m/z 73 m/z 87 m/z 101
73
43
87
no M+.
101
63
Radical site, charge site
• For the interpretation it is assumed in a simplistic approach that

the ion has localized sites for the odd electron (radical site) and
the charge (charge site - McLafferty). In reality the situation is
more complex, where either the radical or the charge site may
drive the reaction
Charge retention, charge +

O
migration +.
O C
CH3
Assuming that upon ionization the C
CH3 CH3 retention
charge remains localized at a
specific site, fragmentation may CH3+
either lead to charge retention or
migration
charge migration
64
Sigma (σ) bond cleavage
• If upon ionization the electron is removed form a single (σ)
bond, cleavage of this bond is favoured. This is e.g. a typical
fragmentation with ionized alkanes.
• Example – 2,2-dimethylpropane
CH3-C(CH3)3 +e- → CH3•+C (CH3)3 → +C (CH3)3 + CH3•
57
H 3C C(CH 3 )3
57
29 41
no M+.
65
Sigma (σ) bond cleavage
n-decane – C10H22
fragments that can stabilize the
charge better are preferred
ie 30 > 20 > 10
3,3-dimethyloctane – C10H22
**
+
m/z113*
*
m/z71**
66
Alpha (α) cleavage (radical site initiation)
• α- cleavage (radical site initiation) arises from the strong tendency

for electron pairing: the odd electron is donated to form a new bond
to the adjacent atom. This leads to the cleavage of the bond next
to the α-atom
.+ . +
CH3 CH2 O C2H5 CH3 + CH2 O C2H5
• The tendency to donate electrons to the adjacent bond increases

in the following order: N > S, O, π, R• > Cl, Br > H.
• α- cleavage is thus particularily pronounced with amines, but also

observed with ethers, ketones, olefines, alkyl substituents, while is
hardly observed with halides
67
General Procedure for the Interpretation of a
Mass Spectrum
1. Test for molecular ion identity: must be the highest peak in

spectrum with even m/z value, if C, O, S, H, Cl, Br, and logical
neutral losses
2. Use isotopes (13C, 34S, 37Cl, 81Br) to help deduce the
elemental composition
3. If high resolution is available, deduce elemental composition
4. Ring plus double bond
5. Fragmentation pattern: important low mass series and primary
neutral losses from M+.
6. Library spectra (NIST, Wiley etc)
68
Q: Assuming these ions are molecular species and were acquired under accurate
mass conditions, which is the correct molecular formula?
C6F12O
C22H46
309.1244
100
C15H20N3O2Cl
C23H32N5O11S2
%
311.1244
310.1244
312.1244
0
100
315.9757
%
316.9757
0
100
310.3600
%
311.3600
100
310.0843
%
310.5843
311.0843
0 mass
308 309 310 311 312 313 314 315 316 317 318 69
?
70
Chemical Ionization (CI)
• Positive Ion Chemical Ionization (Munson and Field, 1966)
– Protonation by ion–molecule reaction or charge exchange

(CE)
– Reactant gas at ~ 0.1-1 Torr ionized by electron ionization
→ reactant ions formed by ion-molecule reactions
→ low energy electron transfer reactions (CE)
– Analytes mixed in ion source with reactant gas

Analyte : reactant gas = 1 : 102 – 104
– Ion source tight (higher pressure) to promote ion-molecule

reactions
1
Chemical Ionization (CI)
• EI produces ions which are generally high in internal energy resulting in
a large degree of fragmentation and in some cases prohibits the
detection of the molecular ion.
• If the pressure of a reagent gas (CH4 or NH3) in the ion source is

sufficiently high (0.5 - 1 mbar) so that many collisions occur within the
residence time of ions inside the source, then reactions may take place
• This is called chemical ionization, a technique whereby ions may be

produced with much less internal energy (sometimes with a known
amount) and less fragmentation occurs.
• The CI process most often yields even electron ions such as (M+H)+ or
(M-H)- which fragment is different ways to odd electron ions. 2
The Source
EI Source CI Source
Filament S S
Filament
N N
Sample Sample
in in
e-
Repeller M+. Repeller e- [M+H]+
Trap Trap
S S
N N
Source pressure ~ 10-6 Torr Source pressure ~ 0.1-1 Torr

3
Positive Ion Chemical Ionization
Formation of reagent ions depends on pressure and residence time:

Methane:
CH4 + e- CH4+. + 2e-
CH4+. + CH4 CH5+ + CH3.
CH3+ + CH4 C2H5+ + H2
CH5+ + M MH+ + CH4
Ammonia:
NH3 + e- NH3+. + 2e-
NH3 + NH3+. NH4+ + NH2.
NH4+ + M MH+ + NH3
NH4+ + M [M+NH4]+
4
NH3CI – Formation of Reagent Ions
NH3+. NH4+
NH3+.
mid pressure – ion/molecule

Low pressure – EI conditions
reactions initiated
NH4+
high pressure – CI conditions

The effect of ion
source pressure is
clearly seen!
NH3NH4+
(NH3)2NH4+
5
NH3CI – Formation of Reagent Ions
NH4+ NH4+
Short residence time Long residence time
NH3+.
NH3NH4+
(NH3)2NH4+
•In these experiments, NH3 pressure is constant

•Residence time is changed by increasing the voltage on the repeller
6
Reagent Ion Intensity vs Source Pressure
+
CH +
4 CH
5
CH4+. CH5+
Intensity
Ion Intensity
0.1 0.5 1.0
Source pressure(torr)
ln(Source Pressure)
(torr)
7
Chemical Ionization
• There are 4 general pathways to form ions from a neutral
analyte M in CI:
M + BH+ MH+ + B proton transfer

M+B [M-H]- + BH+
M + X+ MX+ electrophilic addition
M + X+ [M-A]+ + AX anion abstraction
M + X+. M+. + X charge exchange

M + X-. M-. + X
And others eg anion attachment (Cl-) 8
Thermochemical Considerations
• The tendency of a molecule M to accept a proton is

quantitatively described by it’s proton affinity (PA):
B + H+ BH+
-ΔHr0 = PAB eg CH4, NH3
It is the negative of the enthalpy change in the gas-phase

association reaction between a proton and the neutral
9
Positive Ion Chemical Ionization
Reagent Gas Reagent Ion PA (kcal/mol)

Hydrogen, H2 H3+ 101
CH5+ 128
Methane, CH4
C2H5+ 161
Water, H2O H3O+ 170
Methanol, CH3OH CH3OH2+ 182
Acetonitrile, CH3CN CH3CNH+ 187
Isobutane, i-C4H10 C4H9+ 194
Ammonia, NH3 NH4+ 202
Methylamine, CH3NH2 CH3NH3+ 211
10
• In the subsequent CI experiment where:
M + BH+ MH+ + B
protonation will occur as long as the process is exothermic ie

PAB < PAM
This exothermicity can result in fragmentation of the MH+ ion:
Eint(M+H)+ = PAM - PAB
11
• Protonation under CI conditions
AB + H+ → ABH+
ΔHRx = -PA(AB)
= ΔHf(ABH+) – ΔHf(AB) – ΔHf(H+)
Rearranging yields:
ΔHf (ABH+) = ΔHf (AB) + ΔHf (H+) – PA (Proton affinity)
12
• In positive chemical ionization the reactant gas and the

analyte molecule compete for the proton
• Only if the proton affinity of the analyte molecule is higher
than that of the reactant gas protonation can occur
• In an ideal case the difference in proton affinities is
transferred as internal energy to the analyte molecule during
protonation
• By proper choice of the reactant gas the ionization can be
tailored in such a way that a minimum of energy is
transferred to the analyte
• → can yield soft ionization with little fragmentation
13
Example:
Protonation of ethylamine (PA = 208kcal/mol)
• With methane (PA = 128kcal/mol)
excess energy = 208 – 128 = 80kcal/mol → strong fragmentation
• With methylamine (PA= 211)

excess energy = 211 – 208 = 3kcal/mol → little fragmentation
• If ammonia is used for CI of analytes their PA‘s determine whether a

[M+H ]+ or [M+NH4 ]+ is formed:
• PA (NH3) ~ PA (analyte) → [M+NH4 ]+ and/or [M+H ]+
• PA (NH3) < PA (analyte) → [M+H ]+
• PA (NH3) > PA (analyte) → [M+NH4 ]+ or analyte not observed
14
Comparison of EI, CH4CI and NH3CI
for mw=340 species
EI
no M+.
CH4CI
[M+H]+
NH3CI [M+NH4]+
15
Charge Exchange Chemical Ionization
• The interaction of a positive ion with a neutral can lead to

charge exchange:
X+. + M M+. + X
ΔH = IE(M) – RE(X+.)
• This reaction will occur if the recombination energy (RE) of
the reactant ion (X+.) is greater than the ionization energy of
the neutral M ie if the reaction is exothermic.
• Results in the formation of a radical cation (M+.) where the
excess internal energy can be controlled by selection of X!
16
• Also results in some degree of selectivity as if the IE of M is

higher than the RE of X, then no electron transfer will occur
and M will not be observed
• Reactions of the type:

A-. + B B-. + A
ie formation of a negative ion can also occur provided the
electron affinity of B is greater than the electron affinity of A.
• Rarely used in practice (+ve or –ve CECI)
17
Ion Recombination Energy (eV)

Ne+. 21.6
Ar+. 15.8
N2+. 15.3
Kr+. 14
CO+. 14
CO2+. 13.8
Xe+. 12.1
CS2+. 9.5-10
C6H6+. 9.3
18
For example:
•if the IE of M is 11eV, CECI with Ne+. will yield excess

internal energy of ~+10eV extensive fragmentation
•if the IE of M is 11eV, CECI with Xe+. will yield excess

internal energy of ~+1eV little or no fragmentation
•if the IE of M is 11eV, CECI with C6H6+. will yield excess

internal energy of ~-1.5eV M will not be ionized
19
Negative Ion Chemical Ionization (NCI)
• Reactant Ion Negative Chemical Ionization

– Mixture of N2O and methane forms [OH]-
– M + [OH]- → [M – H]-
• that is, proton abstraction
– CH2Cl2 or CHCl3 forms Cl- by dissocaitive electron

capture
• Proton abstraction [M-H]- from very acidic compounds
• Cl- attachment ion [M+Cl]- formed with acidic
compounds
20
Negative Ion Chemical Ionization (NCI)
• Electron Capture Negative Chemical Ionization

– 3 mechanisms of ion formation:
– M + e- M-. Resonance electron capture (0-2eV)
– M + e- [M-A]- + A. Dissociative electron capture (>2eV)
– M + e- [M-B]- + B+ + e- Ion pair formation (>10eV)
– Generation of thermalized electrons (0 to 2eV kinetic energy) by

bombardment of reagent gas (methane or ....)
– Very soft ionization
– Negative ion yield increases with increasing electron affinity
– Introduction of groups with high electron affinities by derivatization
such as pentafluorobenzyl derivatives
21
Summary
• CI
– Analtye ions must be volatile (same as EI)
• naturally, by sublimation or by derivatization (non-ionic)
– Volatility demand leads to small (<1kDa) molecules
• heating biopolymers destroys them
• extensive derivatization cumbersome
• Desorption techniques minimize any thermal input to the analyte
(DCI)
sample CI source CI source
sample
heat
Direct insertion Desorption
22
Field Ionization (FI)
• Ionization mechanism
– If atoms or molecules are exposed to very high electric fields

(107 –108 V/cm) near a metal surface, a valence electron of the
atom or molecule may tunnel into the anode (+vely charged)
– this is termed Field Ionization, FI (Inghram and Gomer, 1955;
Beckey, 1959)
– Such high electric fields are produced if a voltage of 3 –10 kV
is applied to a fine metal tip (emitter) of < 1 µm radius of
curvature opposite to a counter electrode
– During ionization hardly any excess energy is tranferrred to the
ion “soft ionization“ that is, little or no fragmentation
– Usually forms M+. but also [M+H]+ possible and M-.
1
Field Ionization (FI)
• Ionization
– Much higher ion yields can be generated if a large number of

tips are generated on a thin metal wire (3 – 10 µm i.d.) by
creation of whiskers (dendritic microneedles) by polymerization
of a suitable organic compound (benzonitrile), a process
termed activation C whiskers
– As in electron impact (EI) and chemical ionization (CI) the

sample is introduced to the emitter via the gas phase
– GC, direct insertion probe, heated inlet
– Very polar, non volatile compounds can not easily be introduced
– Poor sensitivity due to low probablity of desorbed neutral
coming close enough to the emitter to be ionized
2
Field Ionization: Activated Emitter
3
courtesy of Springer 2004
Field Desorption (FD)
• In field desorption mass spectrometry (FD-MS) the sample is
dissolved in a suitable solvent and applied directly to the
emitter. By applying high voltage to the emitter (ca. 104 V/cm)
and simultaneously heating the emitter even compounds of
low volatility, such as organic salts, are transferred into the gas
phase
• Yields both M+. and [M+H]+ ions depending on sample polarity
• field desorption was the first mass spectrometric technique
which was suited for the analysis of non-volatile compounds,
such as oligopeptides, oligosaccharides and organic salts.
• Much better sensitivity than FI

4
Field Ionization and Field Desorption
5
Field Desorption – Trityl Chloride
6
Particle Bombardment
Three main techniques:
• Fast Atom Bombardment (FAB) employing 5-10keV Xe

atoms
• Liquid Secondary Ion Mass Spectrometry (LSIMS) using 20-
30keV Cs+ ions
• 252Cf Plasma Desorption – MeV particles created from
radioactive decay of 252Cf
• allowed the MS analysis of high mass (>1000), polar, involatile
and/or thermally labile species – no direct heating of sample
• rely on momentum transfer from fast moving species to sample
suspended in a matrix (FAB/LSIMS) or on a foil (252Cf PD)
1
Fast Atom Bombardment (FAB)
• Fast atoms are produced in a FAB gun.
8KeV 8KeV
Xe+. + Xeo Xeo + Xe+.
That is, a charge exchange process
• Non-volatile molecules are transferred to the gas-phase by bombardment

with a beam of fast atoms (4-10 keV). (Barber et al.,1981) 2
FAB: Mechanism of Ion Formation

3
FAB: Mechanism of Ion Formation

4
FAB/LSIMS of Inorganic Salts
• Clusters of Cs+ with I- forms series

of ions:
– +ve ion MnXn-1 eg Cs5I4+
– -ve ion MnXn+1 eg Cs4I5-
• +ve and –ve ion FAB of CsI

• ions observed up to m/z >12,000
• used for mass calibration in FAB
and LSIMS
5
courtesy of Micromass
FAB/LSIMS: Role of the matrix
The matrix:
• Absorbs primary energy
• Helps to overcome intermolecular forces between analyte
molecules
• Helps to maintain a long lasting sample supply ie replenishes
the damaged surface by diffusion
• Important for ion formation: proton donator or acceptor or
electron donor/acceptor
• Sample must be soluble in the matrix
• Must be a viscous liquid that can survive the conditions in the
high vacuum source to allow extended analysis
6
FAB and LSIMS: Matrices

7
FAB mass spectrum of glycerol matrix
8
1981 – the 1st analytical use of FAB
Peptide – 11mer
Michael Barber et al., J.C.S. CHEM. COMM., 1981

9
Cs+ LSIMS
These spectra represent

state-of-the-art protein
analysis in 1987
B.N. Green et al 35th

ASMS conference - 1987
Note: not m/z! 10

Surface Analysis with SIMS
• SIMS analyzes the secondary ions emitted when a surface is

irradiated with an energetic primary ion beam
• Ar+, O2+, Cs+, O- are formed in a source and accelerated to
very high kinetic energies (keV) and causes the emission of
secondary particles (+ve, -ve or neutral) from the surface
• This beam is rastered across the targets surface
• The secondary ions are subsequently accelerated and mass
analyzed
• This technique is mostly used with solids and is especially
useful to study conducting surfaces.
11
Surface Analysis with SIMS
• High resolution chemical maps ie pictures representing

the chemical distribution of the surface can be produced
by rastering a tightly focused ionizing beam across the
surface.
• Used for trace elemental analysis especially in the
semiconductor and thin coating science
• Excellent technique for the elemental analysis of solid
inorganics
12
Ion Imaging
197Au and 34S signal from a pyrite (FeS2) grain
13
SIMS
Advantages:
• only surface species are ionized

• depth profiling is possible by “milling” away at the sample
with an ion beam
• ions of all elements can be produced
• can be used for small sample quantities
14
SIMS
Disadvantages:
• elemental sensitivity varies (~104) between 10-4 and 10-8 mol L-1
• non-conducting samples will charge up leading to unstable
signals
• isobaric interferences
ie. 56Fe and 28Si2 (m/z 55.9349 and 55.9539)
•this requires a resolution in excess of 5000 to
distinguish by high resolution/accurate mass MS
•a double focusing sector instrument can have

resolutions in the 500 to 10000 range remember,
better resolution is offset by poorer sensitivity
15
Laser Desorption Ionization (LDI)
• laser pulses yielding 106 – 1010 Wcm-2 are focused on a solid

sample surface of about 10-3 – 10-4 cm2.
• The laser pulse ablates material from the surface, creating a
microplasma of ions and neutral molecules.
hυ
eg. CsI(s) Cs(CsI)n+
• LD is used to study surfaces since you can good spatial

resolution with such a small beam of photons
• high sensitivity
• large variation in ionization probability ie. different energy to
vaporize and ionize different samples
1
LDI
• large spread in ion kinetic and internal energies, normally only

fragments of the sample are seen in the mass spectrum due to
dumping so much energy into the molecules, therefore structure is
destroyed
• useful for metals, inorganic salts and some polymers
• signals are very short, therefore LD is best used with a mass
analyzer such as TOF
2
Matrix Assisted Laser Desorption Ionization
(MALDI)
• the compound to be analyzed is mixed in a solvent containing an organic

compound (the matrix) which strongly absorbs at the laser wavelength.
• the solvent is evaporated off leaving the analyte embedded in the matrix material.
• an intense laser pulse deposits large amounts of energy into the sample in a
short period of time.
• this ejects or ablates bulk portions of the solid which contain matrix and analyte
molecules from the surface 3
MALDI
• both UV and IR lasers can be used but most often a N2 (UV@337nm) laser is
used
• little internal energy is transferred to the analyte due to expansion and
evaporation of the matrix material from the analyte so there is little fragmentation
• ionization reactions can occur at any point in the process but the origin of ions
produced in MALDI is not fully understood with numerous possibilities:
•desorption of preformed (M+H)+ and (M+Na)+ ions
•gas-phase protonation 4
•direct photoionization M+. and M-.
MALDI
• among the chemical and physical ionization pathways suggested
for MALDI are: gas-phase photoionization, excited-state proton
transfer, ion/molecule reactions, or desorption of preformed ions.
• the most widely accepted mechanism involves gas-phase proton
transfer in the expanding plume with photoionized or photoexcited
matrix molecules.
• since most matrix materials contain aromatic rings, they can act
as energy gathering chromophores.
M + hυ M*
M* + A AH+ + (M-H)- M = Matrix
A = Analyte
M* + M MH+ + (M-H)-
MH* + A AH+ + M 5
Laser Desorption and Ionization: Mechanism
6
courtesy of Micromass (Waters)
Laser Ionization (without matrix): ion emission
as a function of λ
Absorption spectrum of matrix Absorption spectrum of sample
7
Laser Ionization (with matrix – MALDI): ion
emission as a function of λ
8
MALDI: Matrices
9
MALDI Target - Batch Introduction Process
Bruker Micromass
Can be fully automated!

10
MALDI
MALDI spectrum of an N-linked glycan

11
MALDI–Tof of a monoclonal antibody (m/z ~
150,000)
12
Protein Identification using MALDI
• Large proteins (>100,000 Da) may be multiply charged (doubly or
triply charged): [M+H]+, [M+2H]2+, [M+3H]3+ but no more - very
unlike ESI!
• Smaller proteins and peptides are always singly charged – again,
this is not the case for ESI
• MALDI typically exhibits better sensitivity than ESI because it has
a higher tolerance for other non-peptide sample constituents -
attamole
• MS/MS of [M+H]+ ions provides fewer structurally significant
fragment ions than does [M+2H]2+ ions commonly seen in ESI
• not as useful for peptide sequencing or MS/MS ion database
searching
• Difficult to interface to chromatography
• Also used for synthetic polymer characterization and imaging 13
2D Gel Electrophoresis
14
Protein Identification using MALDI
• Separate proteins on a 2D polyacrylamide gel (2D-PAGE)
• Excise individual spots
• These spots, which may contain 1 or more proteins, are degraded into
small peptides (enzymatically) and measured by MALDI (or ESI)
• The resulting MALDI mass spectrum (primarily [M+H]+ ions) is converted
into a table of molecular weights of the individual peptides present to
yield a Peptide Mass Fingerprint
• Peptide Mass Fingerprint (PMF) or peptide mapping is an ideal method
to identify peptides derived from a protein which are already known and
in a database
• The quasimolecular ions of this mixture of smaller peptides provide a
map or fingerprint which can be searched against protein databases to
provide a protein identification
15
Peptide Mass Fingerprint using MALDI
a .i.
1 50 0 0
1 00 0 0
5 00 0
1 50 0 2 0 00 2500 30 0 0 3 50 0 m /z
16
MALDI-Tof of Polystyrene
(A) mwt~330,000
(B) mwt~600,000
(C) mwt~900,000
17
Reproduced from Schriemer & Li, 1996
MALDI Imaging
Courtesy of S. Khatib-Shahidi, M. Andersson, J. L. Herman, T. A. Gillespie, and R. M. Caprioli. Anal. Chem. 2006, 78, 18
6448-6456.
Inductively Coupled Plasma (ICP) MS
• Inductively coupled plasma used originally and still used today in

emission spectroscopy
• Plasma temperature may exceed 8000oC
• Elements with IP < 10 eV fully ionized → mass spectrometry
• Inorganic samples first digested (e.g with nitric acid), pneumatically
nebulized and introduced as finely dispersed mist into an argon ICP at
+1200 Watt
• Coupled to quadrupole MS and to magnetic sector double focussing
instruments (the latter have much better mass resolution)
• Very sensitive, very specific
• Can suffer from isobaric interferences
1
ICP-MS
2
ICP-MS
• The Ar plasma is generated and maintained at the end of the
glass torch located inside the loops of a water cooled copper load
coil.
• A radio frequency (RF) potential applied to the coil produces an
electromagnetic field in the part of the torch located within its
loops.
• A short electric discharge from a wire inside the torch provides
the electrons to ignite the plasma.
• In the electromagnetic field of the load coil these electrons are
accelerated and collide with Ar flowing through the torch
producing Ar+. ions and free electrons.
3
ICP-MS
• Further collisions cause an increasing number of Ar atoms to be
ionized and result in the formation of plasma.
• The plasma-forming process rapidly becomes self-sustaining and
may be maintained as long as Ar gas continues to flow through
the torch
• These colliding species cause heating of the plasma to ~10,000
K. The high temperatures rapidly desolvate, vaporize, atomize
and ionize the sample. Therefore the sample is turned into atomic
ions which are then mass analyzed
4
ICP-MS
• Each element shows up at its own m/z value, including isotopes
• Intensities are directly proportional to the amount of element
introduced to the torch
• No structural information since complete atomization occurs
• Different ionization efficiencies result in different sensitivity
• Isobaric interferences:
• ArO+ m/z 55.9573 / Fe+ m/z 55.9349

• Ar2O+ m/z 95.9197 / Mo+ m/z 95.9068
• Ar2O++ m/z 47.9599 / Ti+ m/z 47.9479
5
ICP-GCMS – Organotin standards
100.0
80.0
60.0 Sn isotopic distribution

40.0
20.0
0.0
110.00 115.00 120.00 125.00
120Sn+ monitored
12,000
Detection Limit:
1. inorganic Sn – not reported
10,000
ICP-MS Intensity
2. MBT 4.4fg
3. TPrT 5.3fg
4. DBT 9.4fg
8,000 5. MPhT 4.4fg
6. TBT 9.9fg
6,000 7. DPhT 10fg
8. TPhT 11fg
4,000
2,000
0 5 10 15 Retention time (min)
1μL injection of 5ppb standard 6

Atmospheric Pressure Ionization (API)
• conventional ionization methods employ sources that are at high
vacuum (EI, CI, FI/FD, FAB/LSIMS, MALDI) and/or temperature (EI, CI,
FI/FD)
• the introduction of API sources employing a number of different types of

ionization has allowed very robust instruments to be developed for
LC/MS
• These “new” ionization techniques have greatly extended the range of

analytes that can be studied by MS to compounds that are high
molecular weight, thermally labile and polar.
• While the sources are designed to operate at atmospheric pressure we

must still maintain a high vacuum in the rest of the instrument if we want
to perform mass spectrometry!! 1
API Source
reduced pressure
Atmospheric pressure High vacuum
}
}
HPLC inlet Skimmers Lenses
Nebulizer Vacuum Wall
gas inlet
Capillary Octopole
Nebulizer
+
+
+
+
+
+ + + + + + + + + + + + + Mass analyzer
heated N2
Spray is at right angles

to entrance to
MS - orthogonal
Waste Vacuum Pumps 2
courtesy of Agilent
API Source
• High vacuum must be maintained in the mass analyzer and detector
region even though the source is at atmospheric pressure
• The region after the source is heavily pumped with rotary vacuum and
turbomolecular pumps (usually)
• Also, a series of skimmers and flow restrictors are placed between the
source and the mass analyzer region
• These skimmers allow ions to be efficiently transmitted to the high

vacuum region while at the same time allow air, solvent vapours and
other neutral volatile species to be pumped away
• The exact design will depend on the specific instrument type and
manufacturer
3
API Sources
• Electrospray (ESI)
• high flow rate (100μL/min – 1mL/min)
• capillary flow rate (2μL/min - 100μL/min) } pneumatically
assisted ESI
• low flow rate (<2μL/min)
– nanospray (200-500nL/min) – ESI is most sensitive at these
low flow rates
• Atmospheric Pressure Chemical Ionization (APCI)
• Atmospheric Pressure PhotoIonization (APPI)
• Atmospheric Pressure MALDI
4
Relative Applicability of API Techniques
100,000
ESI & APMALDI
10,000
Molecular Weight
1000
APCI &
APPI
nonpolar Analyte Polarity very polar
ESI: Electrospray Ionization & APMALDI

APCI: Atmospheric Pressure Chemical Ionization
APPI: Atmospheric Pressure Photo Ionization 5
EI, CI, GC-MS
Electrospray (ESI)
• Based upon the electrostatic spraying of liquids where a solution is
passed through a needle held at high voltage (kV) relative to a
counter electrode (the entrance to the MS)
• When the solution contains an electrolyte and the needle forms

part of the API source then the fine mist of droplets that emerge
from the needle tip possesses a net +ve or –ve charge determined
by the polarity of the needle and the solution chemistry of the bulk
liquid
• These preformed and then sprayed ions, which are characteristic

of the dissolved analytes, are attracted to the entrance of the MS
by applying appropriate voltages
6
Electrospray (ESI)
• The formation of the spray must be aided by nebulization (pneumatically
assisted) at liquid flow rates higher than a few μL/min
• ions exist in solution, if not, electrospray doesn’t work, it is not an ion

formation technique rather than a technique for extracting ions from the
solution-phase into the gas-phase free of solvent for mass spectral
analysis
• The analyte must be an ion in solution either as a preformed ion such as

or through modifying the solution chemistry to induce a charge
N
• This can be accomplished by changing solution pH or adding cations eg

Li+, NH4+ etc or anions to form adducts eg Cl-, OAc- etc
7
Electrospray Ionization
Charged Droplets Analyte Ions in the gas phase
containing ions in solution - both +ve and -ve
+ +
+ - + Nebulizer assisted >1μL/min
-
+ - + - capillary 2-100μL/min
-
+
-
+ +
+
- normal 0.1-1mL/min
“Classical” - nanospray < 1μL/min

Evaporation
+
+
+ ++ + -- +
+ + Solvent Ion Cluster
Rayleigh + +
--- +
+
Limit +
- -
+ +
+ +
Reached - +
-
+ + +
+
+ Analyte Ion
Coulombic
(proton transfer and
Explosions 8
adduct ions)
The “Source”
High voltage
Power supply electrons
-
- +
+ + +
- + + ++ +
+
- + + +
+ + + + + +
++
- +
-
+
+
- + +
+ +
+ +
+ +
+ +
+
+ +
+ + + +
to MS
+
- + +
+
+
cathode - reduction
Anode -oxidation
+
+
Taylor cone
+
+
+
+
+
+ ++ + + + + +
+ + + + +
+ + + + ++ + + + ++
+
+
+
+
+
+
+
9
Proposed Mechanisms:
1. Charge Residue Model: where the droplet is completely evaporated
leaving “bare’ analyte ions
2. Ion Evaporation Model: field assisted ion desorption

• Requires ~ 107Vcm-1 and a final droplet diameter of 10nm
• Fits well with the observed data
• In either case it is required that the analyte be an ion in solution (+ve

or –ve) or made to be charged by modifying the solution to cause the
analyte to be ionized
• This can be accomplished by changing pH, adding modifiers (Na+, Li+)
10
Electrospray Solution Chemistry
• Mobile phase pH has a major effect for analytes that are ions in solution:
– Basic pH for negative ions
– Acidic pH for positive ions
• Changing pH can enhance performance for analytes that are not normally
ionized in solution
Positive Ion Mode

R1 R1
| |
:N - R2 + HA +HN - R -
2 + A Positive ion mode, [M+H]+
| |
R3 R3
Base Acid Analyte Ion
O O
|| ||
R-C-OH + :B R-C-O- + H:B+ Negative ion mode, [M-H]-
11
Acid Base Analyte Ion
• In the case of acid/base chemistry, ideally we want to be 2 pH units
either side of pK in order to cause complete protonation (+ESI) or
deprotonation (-ESI) to give maximum sensitivity
• In the case of batch introduction (infusion) of sample this is easily

accomplished however in the case when LC is employed it is the nature
of the mobile phase that determines the ions we will observe and the
sensitivity
• For example, in a reversed phase (C18) separation of analytes, in order

to achieve a good separation it is necessary for the analytes to be
neutral in solution so that they may interact with the stationary phase
and achieve a good separation. These neutral species will not yield the
best sensitivity when ESI is used.
12
• Don’t forget, the ESI process is a competition for charge!
• A neutral in solution will pick up charge in a variety of ways and while we

can influence which process is favoured we can not eliminate all
competing ion formation mechanisms
• Not only do proton transfer reactions occur but adduct ion formation is
commonly observed
• Species such as [M+NH4]+, [M+Na]+ and [M+K]+ in positive ion and

[M+OAc]- and [M+Cl]- in negative ion are often observed even though
these modifiers may not have been deliberately added to the solution
containing the analyte
13
+ESI of Nucleotide Homologue (mw=890)
Sample in 1:1 CH3CN/H2O+0.2% formic acid
[M+H]+
[M+Na]+
[M+K]+
[M+NH4]+
14
Electrospray Considerations
Samples:
• Ions in solution: catecholamines, sulfate conjugates, quaternary

amines, carboxylates, phosphorylated compounds
• Compounds that can have a charge induced: carbohydrates
• Compounds containing heteroatoms: carbamates, benzodiazepines
• Multiply charged in solution: proteins, peptides, oligonucleotides
• A curious feature of ESI is the formation of multiply charged ions ie
where z>>1 and sometimes as high as 100
15
Electrospray Considerations
Solution Chemistry Parameters:
• flow rate
• sample pK, solution pH
• solution conductivity
Samples to Avoid:
• extremely non-polar samples: PAHs, PCBs
• Samples containing high levels of buffers/electrolytes as this will
cause ion suppression
Ion Suppression:
• Competition and interference with analyte ionization by other
endogenous matrix species resulting in decreased number of
ions characteristic of the analyte(s) 16
Protein ESI-MS
• In this mass spectrum, each peak represents the quasi molecular ion of
the protein with one more charge attached, usually, but not always, a
proton (H+) eg m/z 942.6 is the [M+18H]18+
• Consequently, each peak can be used to calculate the mwt of the protein
and the resulting values averaged across all charge states.
• This results in mass accuracies for protein mwt determination of + 0.01%
17
or better depending on the type of mass spectrometer employed.
Protein ESI-MS
• Let the unknown mass of the protein be M and the # on charges be n
corresponding to the addition of (M+nH)+
• For 2 adjacent measured masses m1 (high mass) and m2 (low mass)
we can write 2 equations:
m1 = (M+n) (i) and m2 = (M+n+1) (ii)
n (n+1)
Solving for n:
for the ion at m/z 998.0 (m1) = (M+n) 998n = M+n
n
for the ion at m/z 942.6 (m2) = (M+n+1) 942.6n+941.6 = M+n
(n+1)
Consequently: 998n = 942.6n+941.6 n =17 for m1 (m/z 998)
Substituting n=17 in (i) gives M = (m1n)-n = (998x17)-17 = 16,949
• These laborious calculations can be performed for all ion in the distribution or
a software deconvolution can be performed
18
+ESI of a ~39kDa Protein - Infusion@1μL/min
[M+33H]33+ [M+32H]32+
100
100
%
0 m/z
1150 1160 1170 1180 1190 1200 1210 1220 1230 1240 1250
%
[M+50H]50+
[M+22H]22+
[M+18H]18+
0 m/z
200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800
19
Software Deconvolution
Software manipulation of the full scan +ESI data to show protein mwt
39,643+1.3
100
%
0 mass
39300 39400 39500 39600 39700 39800 39900 40000 40100
20
• the charge states of the gaseous ions
pH=2.6 generally represent the charge states in the
condensed phase. These are sometimes
modified by ion/molecule collisions. Ions
such as large biomolecules are highly
charged.
pH=3.0 • the transfer of ions to the gas phase is not
an energetic process. Ions are cold, in fact
the desolvation process further cools ions.
• non-covalent interactions can be preserved
when the species enters the gas phase. This
is significant for the application of ESI to the
pH=5.2 study of biological molecules such as
proteins.
ESI mass spectra of bovine

cytochrome c 21
Raffinose – trisaccharide, mwt=504 +ESI
m/z 522 (M+NH4)+
100
in 1:1 MeCN/H2O+5mMNH4OAc
%
0 m/z
100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600
100
In 1:1 MeCN/H2O+0.2%FA m/z 505 (M+H)+
m/z 522 (M+NH4)+
%
0 m/z
100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600
100
m/z 511 (M+Li)+
+LiOAc
%
22
0 m/z
100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600
Raffinose – mwt=504 +ESI vs -ESI
In 1:1 MeCN/H2O+0.2%FA
m/z 505 (M+H)+
100
m/z 522 (M+NH4)+

%
0 m/z
100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600
100 m/z 503 (M-H)-

m/z 549 (M+HCOO)-
%
0 m/z
100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 23
Not Always Protonated! decamethylferrocene
EI M+. Fe
100
+ESI in MeCN M+.
0 m/z
+ESI in 1:1 MeCN/H2O+0.2%FA 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440
100
M+.
ie no (M+H)+ observed!
%
Electron transfer dominates

0
80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440
m/z
Oxidation 24
ESI – a MS Revolution
• Electrospray ionization (ESI) has allowed mass spectrometry to
investigate a huge diversity of molecules that were very difficult or
impossible to study by MS previously
•proteins, DNA, RNA, oligonucleotides

•polymers, non-volatile inorganic and organometallic molecules
and salts
• As a result it has completely revolutionized mass spectrometry.
• It has also revolutionized the sales of mass spectrometers as the can be

considered to be an analytical technique for biochemistry (big $$).
• Also, it has spurred the growth of more sensitive and exotic types of MS
and combinations of MS analyzers.
25
Atmospheric Pressure Chemical Ionization
(APCI)
• gas phase chemical ionization (CI) process where the vapourized LC
mobile phase acts as the CI reagent gas to ionize the sample
• Mobile phase and analyte are first nebulized (N2) and vapourised by
heating to 350-550oC
• The resulting vapour is ionized using a corona discharge (source of

electrons)
• Subsequent ion/molecule reactions (CI) then cause ionization of the

analyte
• Unlike ESI, analyte ions do not need to exist in solution
• Unlike ESI, best sensitivity is achieved at high liquid flow rates ie 200μL –
1mL/min therefore easily interfaced to conventional HPLC
26
• Analytes must be thermally stable and “volatile”
APCI
27
APCI Process
Analyte containing aerosol
+ ++ + +
+ ++ +
+ + +
Heat and N2 to
+ +
aid volatalization + + ++ Charge transfer to
+ ++ ++ analyte eg H+ transfer,
charge exchange etc
Vapour
Charged reagent
gas formed
needle +
+
+ +
+
Analyte ions
kV corona discharge
- a robust source of e-
28
APCI Considerations
Samples:
• Compounds of intermediate mwt and polarity: PAHs, PCBs, fatty
acids, steroids, phthalates.
• Compounds that don’t contain acidic or basic sites (e.g.
hydrocarbons, steroids, alcohols, aldehydes, ketones, and esters)
• samples containing heteroatoms: ureas, benzodiazepines,
carbamates
• samples that exhibit a poor electrospray response, that is, APCI
can be considered to be complimentary to ESI
29
APCI Considerations
Solution Chemistry Parameters:
• less sensitive to solution chemistry effects than ESI – ion
suppression not so important
• Best sensitivity at higher flow rates than ESI
• accommodates some non-polar solvents not compatible with
ESI (hexane, CH2Cl2 etc)
Samples to Avoid:
• thermally labile, polar and high mwt compounds due to the
vaporization process
30
APCI Mechanism
S + e- → S+. + 2e-
• Solvent molecules are ionized (S+.)
• the solvent is usually a complex mixture of H2O, CH3CN/CH3OH and
mobile phase modifiers
S+. + S → [S+H]+ + S[-H]

• S+. abstracts a hydrogen atom ie a CI process
[S+H]+ + M → [M+H]+ + S
• [S+H]+ ionizes analyte M by proton transfer or proton abstraction
S+. + M → M+. + S
• charge transfer can also occur with solvents like CH2Cl2
31
Atmospheric Pressure Photo-Ionization (APPI)
• Experimentally, you can view APPI as an APCI source where the
corona discharge has been replaced with a Kr lamp
• The 1st step is complete vapourization of the mobile phase used

in the LC separation employing nebulization (N2) and heating to
350-550oC
• gas phase photoionization process

• where the vapourized mobile phase may be photoionized to
form a CI plasma
• or a modifier (dopant) is added to aid the photoionization
process and formation of the CI plasma
• or the analyte can be directly photoionized by photons from
the Kr lamp 32
Atmospheric Pressure Photo-Ionization (APPI)
• It is ionized by high energy photons from a Kr lamp (usually)
causing either direct or indirect (dopant) photoionization
• Very useful for non-polar analytes that are difficult to ionize with
ESI or APCI such as PAH’s
• Unlike ESI, best sensitivity is achieved at liquid flow rates

around 200mL/min therefore easily interfaced to conventional
HPLC
33
APPI Process
Analyte containing +
aerosol +
+
+
+
+ +
Photon ionizes
hυ analyte - Direct + +
Evaporation
+
+ + Analyte ions
+ + +
+ + +
hυ +
Vapour + + +
+ + + + +
+ + + +
+ +
Dopant
added Dopant is photoionized and
acts as reagent gas –
Indirect
34
APPI Mechanisms
Direct APPI:
M + hν → M+. + e-
Analyte molecule M is ionized to molecular ion M+.
– If analyte ionization potential is below Kr lamp photon energy
Subsequently:
M+. + SH → [M+H]+ + S•
Molecular ion M+. may abstract a hydrogen to form [M+H]+
ie a CI process
35
APPI Mechanisms
Dopant APPI:
D + hν → D+. + e-
• Photoionizable dopant D is in excess & yields many D+. ions
D+. + M → → [M+H]+ + D
• Analyte M ionized by proton transfer from dopant or solvent
D+. + M → M+. + D
• D+. ionizes analyte M by electron transfer ie charge transfer
36
Energetics for Photoionization
PhotoMate™ lamp Dopant Ionization Potentials
Krypton 10.0 eV, 10.6 eV Toluene 8.82 eV
Acetone 9.70 eV
Ionization Potentials (IP)
Anthracene 7.4 eV
Solvent Ionization Potentials
Fluoranthene 7.8 eV
Methanol 10.85 eV
Caffeine 8.0 eV
4-Nitrotoluene 9.5 eV Acetonitrile 12.19 eV
2,4,6-Trinitrotoluene 10.59 eV Water 12.61 eV
• The photons from the Kr lamp can only photoionize compounds of lower IP
• Common HPLC solvents like H2O, CH3OH and CH3CN are NOT ionized and
therefore cannot aid ion formation
• In this circumstance, only direct photoionization of the analyte can yield
characteristic ions such as M+. (not very efficient)
– Subsequent ion/molecule reactions can form [M+H]+
37
• Dopants are used that will be ionized by the Kr lamp
Atmospheric Pressure Ionization Techniques
Electrospray (ESI)
• Volatility not required
• Preferred technique for polar, high mwt, thermally labile
analytes
• Ions formed in solution
• Can form multiply charged ions
APCI/APPI
• Some volatility required
• Analyte must be thermally stable
• Ions formed in gas phase
• Forms singly charged ions only 38
Ionization of Analytes
How do we choose which technique to use?

– is the analyte volatile?
– is the analyte thermally labile?
– Does the analyte have heteroatoms that can accept (N > O) or
lose (O >> N) a proton?
– accepts a proton - use positive ion mode
– loses a proton - use negative ion mode
Ion Suppression?
– Dirty matrix would favour the use of APCI/APPI rather than ESI
because they are more tolerant to matrix effects than ESI
39
Chromatographic Considerations
ESI:
• Concentration dependant
– smaller i.d. column gives better sensitivity - nanospray at 200-
500nL/min
• However also works well from 1µl/min to 1 ml/min
• Post-column addition can be used to adjust ionization chemistry
APCI/APPI:
• Mass flow dependant
– column i.d. has little effect on sensitivity
• Works well from 100 µl/min to 1.5 ml/min
• Can be used with normal phase chromatography
40
General Mobile Phase Considerations
• Metal ion buffers interfere with ionization
• Surfactants/detergents interfere with evaporation
• Ion pairing reagents can ionize and create a high background
• Strong ion pairing with an analyte can prevent the analyte from
ionizing
• Some mobile phase additives will cause persistent background
problems
– TEA interferes in positive ion mode (m/z 102)
– TFA interferes in negative ion mode (m/z 113)
41
Mobile Phase Considerations
ESI:
• Solution pH must be adjusted to create analyte ions
– pH 2 units away from pK of analyte
• Organic modifier (CH3OH/CH3CN) has little effect on ionization
• Volatile buffer concentration should be <25mM
• Non-volatile buffers should be avoided or their concentration
should be very low <<5mM
• Na+ and K+ adducts commonly occur
42
Mobile Phase Considerations
APCI/APPI:
• Organic solvent should be a good charge transfer reagent
– use methanol instead of acetonitrile
– proton affinity of CH3OH (182kcal/mol) vs CH3CN
(187kcal/mol)
• Chlorinated solvents can aid ionization in negative mode
• Volatile buffer concentration should be <100 mM
• Non-volatile buffer concentration should avoided or be very low
<<5mM
• Ammonium adducts may occur with ammonium salt buffers
• APPI may require a dopant (eg acetone)
43
Mass Spectra of Prednisolone in Negative
Mode APCI
395.3
with CH2Cl2 Abundance
no CH2Cl2
Abundance
[M+Cl]- 600000
600000
OH
500000
500000
HO O
400000 400000 OH
300000 300000
O
200000 200000
377.3
335.3
365.3
421.3
100000 100000
0 0
150 200 250 300 350 400 m/z 150 200 250 300 350 400 m/z
Prednisolone does not normally ionize in negative mode APCI. In the

presence of CH2Cl2, a very intense [M+Cl]- ion is formed.
44
Mass Spectra of Curcumin in Negative Mode
APCI
with CHCl3 no CHCl3

367.0 367.1
O
90000 [M-H]- 90000 [M-H]-
80000 80000 O
70000 70000
O
60000 60000
OH
50000 50000
40000 40000 O
30000 30000 HO
337.0 337.1
20000 20000
10000 217.1 307.1 10000 191.1 307.1
160.9
0 0
100 200 300 m/z 100 200 300 m/z
Curcumin is an example of a phenolic compound that ionizes equally

well in the presence of oxygen or CHCl3.
45
Caffeine
ESI APCI APPI
195.1
140000 Max: 13143 140000 Max: 71549 140000 Max: 148840
120000 120000 120000
100000 100000 100000
[M+H]+= 195 [M+H]+= 195 [M+H]+= 195

80000 80000 195.1 80000
60000 60000 60000
40000 40000 40000

217.1
196.1
195.1
196.1
20000 20000 20000

121.2
103.2
0 0 0
200 400 600 800 m/z 200 400 600 m/z 200 300 400 500 600 m/z
courtesy of Agilent
46
Methomyl
ESI APCI APPI

347.1
185.0
163.1
Max: 206617
90000 Max: 3663 Max: 95891
90000 90000
80000
80000 80000
70000
70000 70000
60000
[M+H]+= 163 60000 [M+H]+= 163 60000 [M+H]+= 163
50000
50000 50000
40000
348.1
40000 40000
165.1
30000
30000 30000
163.1
20000
106.1
20000 20000
164.1
186.1
163.1
10000
10000 10000
0
0 0
200 400 600 800 m/z 200 400 600 800 m/z 200 400 600 800 m/z
courtesy of Agilent
47
Budesonide
ESI APCI APPI
431.2
453.2
140000 Max: 161681 140000 Max: 78432 140000 Max: 140093
120000 120000 120000
100000 100000 100000

[M+H]+= 431 [M+H]+= 431 [M+H]+= 431
413.2
80000 80000 80000
121.2
60000 60000 60000
431.3
431.3 413.2
40000 40000 40000

323.2
413.2
341.2
103.2
20000 20000 20000

395.2
0 0 0
200 400 600 800 m/z 200 400 600 800 m/z 200 400 600 800 m/z
courtesy of Agilent
48
Sample Matrix Effects
The MS hardware is robust and tolerates non-volatile

components
however…
The ionization process is effected by the concentration and

type of salt/buffer and results in “Ion Suppression” and is
much more prevalent in ESI
“Competition and interference with analyte ionization by other

endogenous matrix species resulting in decreased number of
ions characteristic of the analyte(s)”
49
Sample Matrix Effect in ESI
Composition of HBSS:
Component g/L
Sodium chloride 8
Calcium chloride 0.1
Sulfachloropyridazine (mwt=284) Potassium chloride 0.4
dissolved in water vs. Hanks Balanced Potassium phosphate monobasic
Magnesium sulfate
0.06
0.1
Salt Solution (HBSS) Sodium bicarbonate 0.35
Sodium phosphate dibasic 0.048
Glucose 1
Phenol red 0.011
mAU UV wate
r
wate
r
wate
r
in HB
S S
in HB
S S
in HB
S S
20 in in in
lfa lfa lfa lfa lfa lfa
su su su su su su
10
150000
TIC
100000
50000
Scan mode
4000
EIC, m/z 285 Signal suppression!
2000
0 1 2 3 4 min 50
courtesy of Agilent
Adapting Existing LC Methods to LC/API-MS
• Replace non-volatile buffers with volatile buffers at a concentration

of <10 mM for ES or <100 mM for APCI.
• Substitute phosphates, sulfates, and borates with ammonium

acetate or formate, trifluoroacetic acid (TFA), heptafluorobutyric acid
(HFBA), tetrabutylammonium hydroxide (TBAH)
• If a non-volatile buffer must be used, use a buffer where only
the anionic or cationic part is non-volatile, i.e. ammonium
phosphate, not sodium phosphate.
• Keep the pH the same using volatile additives:
Formic acid, acetic acid, TFA, ammonium hydroxide
• Volatile ion pair reagents should be employed such as HFBA 51

Summary of Ionization Methods
Compound volatile or semivolatile:
• Electron impact (EI):
• M+• and perhaps substantial fragmentation
• Chemical ionization (CI):
• Positive chemical ionization, [M+H]+ (soft ionization - little fragmentation)
• Negative chemical ionization (electron capture), [M]-. (soft ionization - little
fragmentation, can be very sensitive)
• Field Ionization (FI):
• M+•, (soft ionization - little fragmentation)
Compounds non-volatile, methods difficult to couple to HPLC:

• Field Desorption (FD):
• [M+H]+, [M+Na]+ (soft ionization - little fragmentation)
• Fast Atom Bombardment (FAB) and Liquid Secondary Ion Mass Spectrometry
(LSIMS):
• [M+H]+ , [M+Na]+, [M-H]- (soft ionization - quasimolecular ion and fragment ions)
52
Summary of Ionization Methods
Compounds non-volatile, methods difficult to couple to HPLC:

• MALDI: [M+H]+, [M+Na]+, [M-H]- some multiple charging observed (both soft
and hard ionization, quasi molecular ion and fragment ions, biopolymer
analysis)
Compounds non-volatile, methods can readily be coupled to HPLC

• APCI: [M+H]+, [M+Na]+, [M+NH4]+, [M-H]- (soft ionization, low to medium
molecular weight, medium to high polarity)
• APPI: M+•, [M+H]+, [M-H]- (soft ionization, low to medium molecular weight,
medium to high polarity)
• ESI: [M+H]+, [M+nH]n+, [M+Na]+, [M+NH4]+, [M-H]-, [M-nH]n- (soft ionization, low
to high molecular weight, medium to high polarity, biopolymers and organic
salts)
53
Mass Separation
• Magnetic sector instruments

– Single focussing with magnetic sector (B)
– Double focussing with a combination of magnetic and electric
sectors (EB or BE)
• Linear quadrupoles (Q - mass filters)
• Three dimensional quadrupoles (ion traps - IT)
• Linear ion traps (2D)
• Time of flight mass spectrometers (Tof)
• Fourier transform ion cyclotron resonance (FTICR or FT-
MS)
1
Mass Separation and the Lorentz Force
NOTE:
• All mass analyzers function on the basis of the Lorentz Force equation
which describes the force exerted on a charged particle in an
electromagnetic field. The particle will experience a force due to the
electric field (qE), and due to the magnetic field (qvB). Combined they
give the Lorentz force equation:
F = qE+qvB
– F is the force (in newtons)
– q is the electric charge of the particle (in coulombs) = ze
– E is the electric field (in volts per meter)
– B is the magnetic field (in webers per square meter, or equivalently,
teslas)
– v is the instantaneous velocity of the particle (in m/s)
q = ze therefore, F = zeE+zevB 2
Mass Separation: Magnetic Fields (B)
Deflection of ions in magnetic fields:
an ion of mass m and charge z moving with velocity, v, that
traverses a magnetic B at right angles to the direction of the field will
follow a circular path of radius r that fulfills the condition of
equilibrium of FL (Lorentz Force) and centripetal force FC (from the
source accelerating voltage)
FL = qvB = mv2/r = FC r = mv/qB
Right hand rule for a +ve charged particle moving through a magnetic field:
Fingers in the direction of B

Thumb in the direction of v
Palm in the direction of the experienced force
3
Mass Separation: Magnetic Fields
This shows the working principle of a magnetic sector - the
radius, r through which the ion will be deflected depends on the
momentum (mv) of the ion ie the magnetic sector is a momentum
analyzer not a direct mass analyzer!
Since the initial kinetic energy of the ions is given by:

zeV = mv2/2 where V is the accelerating potential
And rearranging for v2: v2 = (2zeV)/m (i)
From: zevB = mv2/r we can derive v2 = (zeBr)2/m2 (ii)
Substitute v2 from (i) in (ii) and rearrange: m/z = eB2r2/2V

4
m/z = eB2r2/2V
• Therefore specific values of V and B allow ions unique in

m/z to pass to the detector. Variations in V or B will cause
ions to collide with the walls of the flight tube therefore at any
unique value of V or B only one specific ion will be passed to
the detector. In practice only B scans are preferrred when
generating full scan data over a large (>50Da) mass range
• One exception to this is when high resolution, accurate

mass measurements are made where Vacc scanning is
preferred as voltages can be controlled and measured much
more accuartely than can B 5
LR EI of mw=308 (Resn~950)
PFK
PFK
Resn~9200 308.1085
C16H20O4S1
1ppm error or 0.3mmu
PFK
6
Deflection of ions of different masses in a
constant magnetic field
•This is how Aston’s original mass spectrograph operated!

• In modern instruments, the magnetic field is scanned to bring ions
of different m/z ratios successively to the detector
7
Directional (angular) focusing of a magnetic
field
Divergent ions of the same m/z will be brought into focus by a magnetic field
8
• One significant drawback with employing B scans is that

the initially accelerated ions have a kinetic energy spread
which exhibits itself as increased peak width ie low
resolution.
• To overcome this problem an electric sector (ESA) is
combined with the magnetic sector to produce what is
called a double focusing instrument.
9
Principle of the Electrostatic Sector (ESA)
• Remember the Lorentz Force equation which describes the
force exerted on a charged particle in an electric field
F = qE
• If a radial electrostatic field E is created between 2 curved

plates held at oppositely charged potentials of +E and –E, an
ion of charge z moving with velocity v will traverse this field
when its electrostatic force equals the centripetal force:
zeE = (mv2)/r
And since the kinetic energy of an ion 1/2mv2 = zeV, then
r = 2V/E
Note: the trajectory is independent of m and z and so the ESA

is not a mass analyzer but reduces kinetic energy dispersion
which results in narrower peaks and increased mass
10
resolution
Double Focusing Mass Spectrometers
• Many geometries have been tried however in general

they can be categorized as:
– Normal or Forward geometry where:
Source ESA Magnet Detector
• and:
– Reversed geometry where:
Source Magnet ESA Detector
11
Double Focusing (Nier Johnson):
Reverse Geometry
12
Double Focusing (Mattauch-Herzog geometry)
Double focusing in a plane
→ photo plate
13
Mass Resolution: Definition
δm10%
δmFWHM
10% Valley
• The 10% valley definition: δm = the mass difference between two peaks
which are separated by a valley equal in height to 10% of the height of
the smallest peak
• The full width at half maximum (FWHM) definition: δm = the width of a
peak at half-height
14
Mass Resolution
• The FWHM definition is easier to apply (only need one
peak), but gives a resolution about twice that of the
10% valley definition
• Resolution for sector instruments is usually given as the
10% valley figure.
• High resolution has some obvious advantages:
-It allows one to resolve ions that are isobaric
-The narrower a peak, the easier it is to measure its
position accurately
15
Mass Resolution
• Low resolution: <2,000. Suitable only for nominal mass

measurement.
• Medium resolution: 2,000-20,000. Suitable for accurate mass
measurement. Resolve isotope clusters of high charge states.
• High resolution: >20,000. Better than medium resolution. You
can never have too much resolution!
• In practice, there is a trade-off between resolution and sensitivity.
The ions are not coming from a point source: they exit the
source through a slit of finite dimensions, and cannot be perfectly
focussed. Slits and lens help to compensate for this by cutting
out ions from the centre of the beam and focussing. To get very
high resolution, the slits have to be narrowed, which means that
a lot of ions are lost. 16
How Many Possible Elemental Compositions?
without isotope abundance information 2% isotopic 5% isotopic
accuracy accuracy
Mol 10ppm 5ppm 3ppm 1ppm 0.1ppm 3ppm 5ppm
mass
150 2 1 1 1 1 1 1
200 3 2 2 1 1 1 1
300 24 11 7 2 1 1 6
400 78 37 23 7 1 2 13
500 266 115 64 21 2 3 33
600 505 257 155 50 5 4 36
700 1046 538 321 108 10 10 97
800 1964 973 599 200 20 13 111
900 3447 1712 1045 345 32 18 196
17
How Many Possible Elemental Compositions?
• No difference between high mass accuracy in the

low m/z range and 3ppm mass accuracy and 2%
isotopic abundance accuracy
• Magnetic sector – 1 to 5ppm
• FTICRMS – 0.5 to 1ppm
• Tof – 1-10ppm
• Orbitrap – 1-5ppm
18
“New” Developments in Magnetic Sector
Instruments
• Large, high field magnets
– Mass range up to 10,000 Da at full accelerating

potential (10 kV) for analysis of large biopolymers
– Example: bovine insulin (MW 5734)
• Laminated magnets
– To reduce magnetic hysteresis
– Total cycle time < 1 sec, fast scanning
19
Linear Quadrupoles (2D - mass filters)
20
Linear Quadrupoles (2D - mass filters)
• Four hyperbolic rods (cheap version: circular rods) – compromise!

• Opposite pairs of rods are connected electrically but are of
opposite polarity
• Each pair of rods has a DC (U) + AC (V0 cosωt) Rf voltage applied:
1 pair of rods: -(U + V0 cosωt) and the opposite pair: +(U + V0 cosωt)
where, ω = radial frequency = 2πf
• During a mass scan, the DC and AC voltages are ramped but the
ratio of DC/AC (ie U/V0)is kept constant
• For a given DC and AC amplitude, only ions with a given m/z (or
m/z range) have stable oscillations and are transmitted and can be
detected
21
Quadrupole (end view)
Hyperbolic Round
Equipotential Field Lines
22
Superposition of RF and DC Voltages
Applied to Rods
4000
3000
2000
RF VOLTAGE
6000 V P/P
1000 AT 2500 u
500 dc VOLTAGE
500 V AT 2500 u
VOLTAGE
0
(V)
-500
-1000
-2000 ATOMIC MASS UNITS

(u)
-3000
-4000
23
Ion Motion
DC + DC -
+ve ion X +ve ion Y
Z Z
+ -
AC
AC + -
+ve ion X +ve ion Y
Z Z
+ -
AC+DC
- •This motion is very complex!
•DC fields focus +ve ions in the +ve plane and
+ve ion Y defocus them in the –ve plane
•The superimposed AC helps correct this defocusing
Z effect
- 24
Linear Q: Equations of Motion
From the electrical part of the Lorentz equation, we can derive the
equation of motion (x and y directions) for a particle in a combination
of DC and AC Rf fields the Mathieu equation:
2
d u
+ ( au − 2qu cos 2ξ )u = 0
dξ 2
– u represents the x or y transverse displacement. We do not

consider displacement in the z direction because the electric field is
0 along the asymptotes of the hyperbolic rods.
– The 2 parameters characteristc of the field (a and q) are given by:
8 zeU and − 4 zeV

a x = −a y = q x = −q y =
mr02ϖ 2 mr02ϖ 2
25
Linear Q: Equations of Motion
• Where the variable ξ is the time in radians of the applied field = ωt/2
• U is the DC voltage and V is the AC Rf voltage of frequency ω
• r0 is the radius of the instrument aperture
• Plotting a against q gives the Mathieu stability diagram of the linear
quadrupole field - a/q = 2U/V
• Typical values are:

- U = DC voltage (~200 - 1000V)
- V = AC voltage (~1000 - 6000V, 1-2MHz),
- m = mass of ion, e = electonic charge, z = # of charges on ion
- 2r0 = distance between the rods - 1-2 cm
26
Stability Diagram
27
aq Space
• Note:
– Both +ve and –ve abscissa with a values ranging up
to 10 and q values ranging up to 20
– In practice we only operate in the +ve area of region I
Why?
– Because in order to have a and q values >1 we would
require VERY high DC and AC voltages which is not
practical
28
Stability Diagram
L1, only 1 ion has a stable trajectory all

others ions are lost therefore adjacent
a ions are resolved from each other
0.3
L1
Y unstable
.. L1 = L2
.. L2
L2, 3 ions have a stable trajectory
0.2 Operating lines . . .. at the same time therefore these

3 ions would not be resolved from
..
a/q constant each other
In practice, the ratio of a/q is changed

by changing the DC voltage
0.1
X unstable
X and Y Stable What would happen if no

DC voltage is applied?
0.4 0.8 q 29
Ion Motion
30
Conceptualizing a Q scan
a
0.3
Operating or scan line

stable region of m1
stable region of m2
0.2
m1 < m2 < m3
stable region of m3
0.1
0.4 0.8 q 31
Mass Range and Resolution
• Depends on 5 parameters:
• Rod length (L) – 50 to 250mm
• Rod diameter (r) – 6 to 15mm aligned to μm accuracy
• Maximum supply voltage (Vm)
• AC (Rf) fequency (f)
• Ion injection energy (Vz) - ~5 volts
• From the theory of quadrupole operation the following

relationship can be derived:
Mmax = 7x106Vm/f2r2
Consequently, as r and f increase, Mmax decreases and

as r and f decrease, Mmax increases
32
Mass Range and Resolution
• The resolution limit of a quadrupole is governed by the

number of cycles of the Rf field to which the ions are
exposed: 2
M/ΔM = 0.05 fL m/2eVz
• Consequently, as both f and L increase so does resolution.

If L in increased then f can be decreased and vice versa
• Scanning speeds as high as 6,000 amu/sec and mass

resolution of 10,000 is attainable
33
Linear Q
Advantages:
• Small and light weight ~1 foot long
• Inexpensive
• Simple to operate – complete computer control
• Low accelerating voltage – handles high source pressures better
• Full scan mass spectra and selected ion monitoring (SIM) for
quantitation
Disadvantages:
• Unit mass resolution only and limited mass range
• High mass discrimination
• Rod contamination causes further imperfections in the
quadrupole field – compromises resolution and sensitivity
34
Linear Q
Other applications:
• QQQ for MS/MS
• Hybrid instruments eg BEQQ and QqTof
• Ion lenses (hexapoles and octapoles)
• Collision chambers for MS/MS ie QQQ and BEQQ etc
• Prefilter – before mass resolving rods to reduce
contamination
35
Quadrupole 3D Ion Trap (QIT)
Ion trap consists of three electrodes:
Cap
Cap
r0
Ring
Cap
• ring electrode (hyperbolic shape)

• 2 hyperbolic electrodes - end caps
• Orifice for ion injection
• Orifice for ion ejection
• Pulsed introduction of ions
36
• Ions accumulated in the trap ie “trapped” and then further

experiments can be performed on the trapped population of
ions eg MS or MS/MS or even MSn
• During this time, ions are gated away from the IT and lost
• employs:
- AC (Rf) to trap and scan ions out to detector – no DC
component
- He buffer gas necessary for efficient trapping of ions
directed into the ion trap
z He present at all times therefore no delay between MS and
MS/MS experiments
37
QIT (properties)
• Ion trap volume very small (7mm i.d.)
• High sensitivity (10-18 mol) (scan mode)
• High mass range : 6,000
• Higher mass resolution than Q ~x2-3
• High dynamic range: 106 depending on space charging
• MSn capabilities
• Low mass cut-off is a disadvantage
• Helium is introduced intentionally into the ion trap (10–3 mbar)
– Needed as a buffer to absorb kinetic energy of incoming ions without chemical
interaction so they can feel the effect of the trapping field - dampening
(cooling) of oscillations
– collision partner for MS/MS and MSn
• Ions are concentrated in center of ion trap
• Better resolution and better sensitivity than Q
38
QIT (ion motion)
• Between the three electrode a quadrupole field exists, which
forces the ions to the center of the trap
• The farther the ion is removed from center of trap the
stronger is the exerted electric force
• The ions oscillate within the trap, but with a rather complex
sinusoidal motion
• The ion motion can be described by Mathieu’s differential
equations
39
• For the QIT, the electric field has to be considered in 3
dimensions. The electric field can be descibed by the expression:
Φx,y,z = Φ (r2 - 2z2)
0
r02
• The equations of ion motion in such a field are:
d²z/dt² - (4e/mr0²) [(U - V cos2ωt)z = 0

d²r/dt² + (2e/mr0²) [(U - V cos2ωt)r = 0
• Solving these Mathieu type differential equations yields the

parameters az and qz
az = -2ar = 16eU/(mr0²ω²) and qz = -2qr = 8eV/(mr0²ω²)

Where ω = 2πf, f = fundamental Rf frequency of the trap (~1MHz)
40
QIT (Ion stability diagram)
Endcap
Ring Electrode
q = 0.908
q < 0.908
Ring Electrode
Endcap
41
courtesy of Spektrum Akademischer Verlag
QIT (stability diagram)
• Ions are only stable both in r and z direction for certain

defined values of a and q
• Ions oscillate with so called “secular frequency”, f, which
differs from the frequency of applied Rf field because of
inertia (in addition oscillations of higher order)
• Ions of different m/z are simultaneously trapped, V
determines low mass cut-off at qz = 0.908, which increases
with V
42
QIT (mass selective ion stability scan)
• Mass scan is possible by increasing the amplitude of the

voltage on the ring electrode (U = 0, az = 0 ie no DC
voltage)
• Scan line: While scanning along this line (a=0) ions
become increasingly non stable and exit the stability
diagram at qz = 0.908.
• Trajectory of these ions in z- direction.
• Ions exit from trap through holes in end cap.
• Linear scan function
43
QIT (resonance ejection)
• An additional Rf voltage with low amplitude is applied to end

caps.
• If frequency of this additional voltage is equal to frequency of
oscillating ions, ions take up energy exponentially and
become non stable:
→ Resonance ejection ( at q < 0.908)
• Mass scan with resonance ejection leads to
– Higher resolution
– Higher sensitivity
– Higher mass range
– Faster scanning – up to 26,000 amu/sec
– Improved reproducibility due to higher net ion sampling
44
rate
QIT: Mass Scan – resonance ejection
• 1: Clear Trap
• 2: Accumulation Time
• 3: Scan Delay
• 4: Mass Analysis
Space Charging
~ 300 Ions ~ 1500 Ions ~ 3000 Ions ~ 6000 Ions

524.3 524.4 524.5 524.8
100 100 100 100
Relative Abundance
80 80 80 80
60 60 60 60
525.7
40 40 40 40
525.3 525.5
525.4
20 526.7
20 20 20
526.5
526.3 526.3
527.5 527.5
0 0 0 0
522 530 522 530 522 530 522 530
m/z
Good resolution Poor resolution
and mass accuracy and mass accuracy
46
Courtesy of Agilent
QIT (space charge)
• With increasing number of ions trapped the space charge

increases
• Space charge distorts the electric field
• Deterioration of resolution, sensitivity and mass accuracy
Solution:
Pre-scan or measure in real time to control the
number of ions (or more correctly, the number of charges)
in the trap (a maximum of ~103 - 104)
47
Linear (2D) traps
• Similar idea to 3D traps with a “new” 2D geometry
• Rf only quads with DC voltage end electrodes
• Larger size than 3D IT – higher ion capacity (~x50) therefore
fewer space charge problems
• More than one design for this type of trapping instrument
• Hybrids such as QQQ where Q3 can also be used as a linear
trap and LT-FTICR
48
Trapping Forces in a Linear Ion Trap
Radial Trapping RF Voltage
Axial
Axial
Trapping
Trapping
DC
Voltage
Exit Lens
Radial Trapping RF Voltage
Courtesy of Sciex Resonance Excitation 49
Linear Ion Trap – 2nd design
y
y RF +
DC 1 DC 2 DC 3
RF - RF - x
z
DC 1 DC 2 DC 3
RF +
• For Axial Trapping 3-130 V Radial Quadrupolar Trapping

DC3>DC2>DC1 1.2 MHz 5KV0-P
• To Contain ions:
DC1=DC3>DC2
y
GND
AC+ AC - x
GND
Radial Dipolar Excitation

Courtesy of Thermo 50
5-600 KHz 0-400 Vpp
Linear Ion Trap vs 3D Trap
No low mass cut-off

Trapping Efficiency: >10
Detection Efficiency: doubled
Overall Efficiency: >10
Ion Capacity (Spectral): >20
Scan Rate (amu/sec): 4x
Highly Efficient MSn: 5x over 3D IT
51
Time of Flight (Tof)
Principle:
Ions of different mass (accelerated by the same field, V)
have different velocities and thus flight times. The larger the
mass the slower the ion:
zeV = mv²/2
Ion formation:
Ions are introduced to the Tof in pulses (e.g. MALDI or
orthogonal extraction from a continuous beam such as ESI)
Ion detected by analogue or time to digital converter (GHz
ADC or TDC)
• Linear Tof (high mass range but low mass resolution)
• Reflectron Tof (lower mass range but high mass resolution)
52
Mass Separation: Time of Flight (Tof) MS
acceleration region
(drift region)
53
Basic Principles
Since the initial kinetic energy of the ions is given by:
zeV = mv²/2 (i)
velocity: v = (2zeV/m)1/2 (ii)
time of flight: t = L/v = L[m/(2zeV)]1/2 (iii)
m/z = 2eVt2/L2 (iv)
Example:
For C6H5+. and C7H7+., (m/z 77 and 91), accelerated at 10kV, what are the
velocities of these 2 ions and how long would it take them to traverse a 2m flight
tube?
using eqn (ii) v77 = (2x1x1.6022x10-19x10,000/m)1/2
m(kg) = 0.077/6.022x1023 = 1.279x10-25
v77 = 128,759m/s 15.53μs
similarly for v91 v91 = 118,457m/s 16.88μs
V is the extraction pulse potential (V)

L is the length of field free drift zone (m)
t is the measured time-of-flight of the ion (s) 54
e = 1.6022x10-19C
Example cont
• From eq (iii), difference in flight time:
tA/tB = (mA/mB)1/2
• Consequently, this square root relationship causes Δt for

a given Δm/z to decrease with increasing m/z
• For example:
Δt/amu is calculated to be 114ns at m/z 20
to be 36ns at m/z 200
to be 11ns at m/z 2000
• Tof mass analyzer depends on the ability to accurately

measure these short time intervals to make it a useful MS
55
Linear Tof
• Transmittance as high as 90%
• Ions introduced into the flight tube have a temporal and kinetic
energy distribution which yields relatively poor mass resolution.
• Kinetic energy spread can be reduced by employing Delayed Ion
Extraction
Principle of Delayed Ion Extraction:
• Ions are formed during a short pulse of a few nanoseconds

• The acceleration (extraction) field is only applied after a delay of
some hundreds of nanoseconds:
• At the beginning of the extraction ions with high initial velocities
have traveled further than slower ones. Therefore after the second
extraction pulse they do not experience the full acceleration
potential.
• Thus the initially faster ions will be accelerated less than the initially
56
slower ions.
Reflectron Tof
Same m/z but
different kinetic
energy
• In a reflectron Tof, the ions traverse the drift tube and penetrate into
an electric field (ion mirror) where their direction is reversed.
• Faster ions (with higher kinetic energy) penetrate farther into the
electric field than slower ions (with lower kinetic energy).
• Thus faster ions have a longer flight path and therefore need
approximately the same flight time as the slower ions which have a
shorter flight path.
57
Tof: Advantages and Disadvantages
• Extreme mass accuracy
– reflectron ~ 5-10ppm
– limited with quadrupole MS, poor with ion traps and linear Tof
• High mass resolution
– reflectron ~5,000 to 20,000
– Quadrupole MS, ion traps and linear Tof operate closer to unit mass
resolution at m/z ~ 103
• Extreme mass range
– linear >105 Da, reflectron <104 Da
– Ion traps and quadrupoles are limited to ~6,000 Da
• Acceptable linearity for linear and reflectron Tof
– not as good as quadrupole MS, but similar to ion traps
• Very good scan-to-scan reproducibility for linear and reflectron Tof
– as good as quadrupole MS 58
Fourier Transform Ion Cyclotron Resonance
(FTICRMS – FTMS)
Principle:
An ion of velocity v entering a uniform magnetic field B
perpendicular to its direction will move on a circular path by action of
the Lorentz force, the radius rm is given by:
rm = mv/qB
Upon substitution with v = rmω, the cyclotron angular frequency ωc
becomes:
ωc = qB/m
• cyclotron angular frequency is independent of ions initial velocity
but is a function of it’s mass, charge and the applied magnetic field
• once trapped, the ions oscillate with a cyclotron frequency that is
inversely related to their m/z ratio
59
FTICR MS
• Basic Construction:
– a cell where ions are trapped by intense, constant magnetic field and
applied voltage
– The cell accepts ions in a “pulsed” mode from the continuous ion beam
– Detection of the ions is based on the FT deconvolution of the image
current the circulating ions induce in a pair of detector plates after
excitation with a resonant Rf pulse.
60
FTICR MS cont.
• MS/MS:
Excitation of the ion is achieved using a variety of techniques. Namely:
– Sustained off-resonance Irradiation Collision-Induced
Dissociation (SORI-CID)
– Infrared Multiphoton Dissociation (IRMPD)
– Electron Capture Dissociation (ECD)
– Blackbody infrared radiative dissociation (BIRD)
• High sensitivity and >105 mass resolution

• MS/MSn
• Cyclotron frequencies can be measured with very high accuracy and
precision leading to ultra high resolving power and high accuracy mass
measurements
61
Ion Trapping and FTICR MS
• ions enter the cell (or are created internally) and they begin their cyclotron
motion, orbiting around the centre of the magnetic field
• since the magnetic field is quite high (typical minimum of 4.7T, but this is
increasing) the ions are trapped in the radial (x,y) direction.
• Resolving power and scan speed increase linearly with B 62
Ion Trapping and FTICR MS
• by applying small, equal potentials to the two end or “trapping”

electrodes, the ions are confined in the z or axial direction.
• ions can be confined for very long periods of time such that
ion/molecule reactions or even slow unimolecular dissociation
processes can be observed and monitored.
63
FTICR MS Detection
• In FT detection, all ions, regardless of their mass are detected at the
same time.
Ions before excitation.

They have their natural
cyclotron radius within the
magnetic field.
• Once ions are trapped inside the ICR cell they are excited by a fast
sweep of all the Rf frequencies, exciting the ions to cyclotron motion
with a larger radius.
64
FTICR MS - Detection
Before excitation
After excitation
• All ions are resonantly excited for the same amount of time.
• Each ion retains its characteristic cyclotron frequency (depending on
m/z) but their radii of orbit increase.
• After excitation all ions have the same radii of motion since they were
irradiated with Rf of the same amplitude for the same amount of time.
• Once the Rf is turned off, each ion packet, consisting of ions of the
same m/z value induces an image current on two sets of receiver
plates which are part of the ion cell. 65
FTICR MS - Detection
When a packet of ions (+ve)

approaches an electrode, electrons are
attracted from ground and accumulate
in that electrode causing a temporary
current.
As the ions continue to orbit, the

electrons accumulate in the other
electrode. The flow of electrons in the
external circuit represents an image
current. The amplitude of the current is
proportional to the number of ions in
the packet.
66
FTICR - Detection
Rf Excitation
Time
Detected time domain

image current
Time
Fourier Transform
Resulting mass domain

Spectrum
m/z
• the frequency of the image current oscillation is the same as the frequency of the
ion’s cyclotron motion which is related to mass. A small AC voltage is created
across a resistor and is amplified and detected.
• using FT techniques all ion packets, each containing ions of the same mass, are
detected. The decay of the image current (as the excited cyclotron orbit radius
decays) is detected in time and transformed into a frequency domain signal by 67
a
Fourier transform.
FTICRMS
• Very high resolution is possible. The current record is 8x108,
and routine values are 100,000 or so.
• Long trapping times are possible, allowing for ion-molecule
reactions.
• Good sensitivity.
• Like the ion trap, the FTICR cell works well with pulsed
sources.
• MSn capability
• However, expensive because of the cost of superconducting
magnets and the very high vacuum requirements.
• Difficult to operate
68
FTICRMS
RT:
100
95 Sample: very complex crude extract 78.81
90
85 from human blood platelets

80 86.60
75 Amount: unknown, but very low conc.
Relative Abundance
Flow: 200 nl/min

70
65
Scan Cycle: 1 spectrum every 3.5 s

60
55
50
45
49.33
40
35 72.26
30
25
66.96
20
15
100.26
10
40 45 50 55 60 65 70 75 80 85 90 95 100 105 110

Time (min)
HCT116_A_030523101055 # 2189 RT: 72.26 AV: 1 NL: 7.84E4 HCT116_A_030523101055 # 2189 RT: 72.26 AV: 1 NL: 7.84E4
T: FTMS + p ESI Full ms [ 200.00-2000.00] T: FTMS + p ESI Full ms [ 200.00-2000.00]
100
579.29291 579.29291
100
95
95
R=315540
90
90
85
RP: 400,000 @ m/z 400

85
80
80
75
75
70
70
65
Expand 65
60
Relative Abundance
60
857.44525
Relative Abundance
55
55
50
50
557.31122
45
45 R=323474
40
557.31122
40
35
574.33777
35
R=312472 595.26740
30 30
25 279.15924 R=300047
891.38403 25
20 20
15
301.14130 654.25793 15
10 10
400.23349 1157.58093
5 5
0 0
300 400 500 600 700 800 900 1000 1100 1200 550 555 560 565 570 575 580 585 590 595 600 605
m/z m/z
69
Courtesy of Thermo
FTICRMS Resolution
70
Courtesy of IonSpec
Hyphenation in Mass Spectrometry
• Gas Chromatography – Mass Spectrometry (GC-MS)

• Liquid Chromatography – Mass Spectrometry (LC-MS)
• MS-MS and MSn - Tandem Mass Spectrometry
WHY?
• the ability to interface an orthogonal separation technique to
MS greatly increases the information content that can be
derived from complex mixtures
1
Gas Chromatography – Mass Spectrometry
(GC-MS)
• Both are gas phase techniques although at
somewhat different pressures:
• Ion source normally at high vacuum (EI and CI)
• GC operates at ~ 5-10psi
GC MS
Quite a stable and “friendly” relationship – they just get along

as both are gas-phase processes 2
GC-MS
• Coupling of capillary GC to MS simple, as low carrier gas
flow rates (1-2 mL/min) easily tolerated by MS vacuum
system.
• Carrier gas always helium (low viscosity, low mass).
• Fused silica capillary introduced directly into the ion source

via a transfer line which must be heated.
• Operation modes of a GC/MS:

– Full scan → TIC/RIC
– Selected Ion Monitoring (SIM)
3
Operating Modes
• Full scan
– selective, sequential transmission of ions to the detector in a continuous
fashion (BE, EB, Q, QQQ, 3DQIT)
– qualitative information
• Selected Ion Monitoring (SIM or SIR)
– Mass analyzer jumps from one pre-selected m/z value to the next
– Only the response from the selected ions is recorded – no mass spectra
– Detector time for the selected ions is increased by a factor of 10 – 100 and so
is the sensitivity
– Used for quantitative analyses – target compound analysis
• Example:
– If a Q is scanning from m/z 100 – 1100 in 1sec then each ion is recorded for
1msec
– If the same Q is set to jump between only 4 ions in a SIM experiment then
each ion is recorded for 250msec 4
GC-MS: Full Scan
5
GC-MS: Selected Ion Monitoring (SIM)
6
Liquid Chromatography – Mass Spectrometry
(LC-MS)
Historical approaches:
• There is a basic compatability problem when an LC is interfaced to a MS:
– LC: 25-50oC, 200nL/mim – 1mL/min liquid flow rate at 100-3000psi
– GC: 50-300oC, 1mL/min He(gas) at 5-10psi
– MS: 200oC, 1x10-6mbar (high vacuum) and 20mL/min gas flow
• The vacuum problem – gas load:

• 1mL/min of hexane(l) yields approx 172mL/min gas
• 1mL/min of water(l) yields approx 1240mL/min gas
• Conventional MS can pump ~20mL/min of gas and maintain high
vacuum
• Atmospheric Pressure Ionization solves many of

7
these problems
Liquid Chromatography – Mass Spectrometry
(LC-MS)
• In GC, the “mobile phase“ normally referred to as the carrier
gas is He and is easily removed however in LC the mobile
phase is a complex mixture of solvents which might contain
buffers, pH modifiers and/or ion-pair reagents – much more
difficult to volatalize and remove
• LC is most commonly used for compounds that are not
easily analyzed by GC because they are thermally unstable,
involatile or high mwt
• MS requires species in the gas phase therefore we must
transfer these difficult compounds into the gas phase without
chemical or thermal modification
8
LC-MS: a difficult courtship
MS
Clearly defined boundary

(picture courtesy of P.J. Arpino)
LC
• It is the function of the interface to “blur” the distinction between

the gas and liquid phase as far as the MS is concerned
• Q: does the fish fly or does the bird swim?
• A: a good question – compromise! Perhaps the bird doesn’t
realize it is swimming and the fish doesn’t realize it’s flying! 9
LC-MS
Why LC-MS?
• LC is capable of providing separation of compounds unsuitable for
GC (even with derivatization)
• Polar, ionic, involatile, high mwt and thermally labile analytes are
readily chromatographed by LC
• Sample clean-up can be more straight forward for LC
• LC allows selection of stationary and mobile phase
• Conventional detectors (UV, FL etc) exhibit good detection limits
but have limited specificity
• The MS is a universal (sometimes), sensitive and highly specific
detector
HOWEVER:
• A GC column is much more efficient than an LC column ie
HETP 10
Solutions to the Problem:
• There are 2 basic approaches to the problem of interfacing an LC to a
MS:
– The “spray” type interfaces which introduce all or a portion (splitting) of
the LC eluent to the source :
– Direct Liquid Introduction (DLI)
– Thermospray (TSP)
– Continuous Flow FAB (CF-FAB)
– Atmospheric Pressure Ionization (ESI, APCI and APPI)
– The “enrichment” type interfaces which preferentially remove the LC

solvent from the less volatile analytes before introduction to the
source:
– Moving Belt
– Particle Beam
• This is by no means an complete list but describes the most important11

Direct Liquid Introduction (DLI)
• Liquid is introduced directly into the high vacuum source

– 10 – 40μL/min of liquid
– Sprayed through a 2-5μm laser drilled hole – prone to blockage
– Desolvation chamber vapourizes analyte and liquid
– Vapour enters CI ion source
– Proton transfer and proton abstraction dominates
– Only CI type spectra with mobile phase forming the CI plasma
– Only volatile buffers can be used
12
– Works well for reversed and normal phase solvent systems
Thermospray (TSP)
• Capillary is heated to partially vapourize the mobile phase supersonic jet of

vapour and liquid
• Mobile phase @ 1mL/min must contain a volatile buffer - normally NH4OAc
• Reversed phase only ie solubilize the NH4OAc
• Ionization occurs through ion/molecule reactions with either NH4+ or OAc-
• Resulting mass spectra are NH3CI like in +ve mode
• Much more robust than DLI but classical TSP limited to reversed phase separations
• This can be overcome to some extent by employing an alternative form of ionization
such as a discharge voltage (APCI like) then termed Plasmaspray or a heated
13
filament as in a conventional EI/CI source (fragile)
Continuous Flow FAB
14
Continuous Flow FAB
• Analyte introduced continuously to the probe tip at 5 –

10μL/min and normally contains a small amount of a matrix
eg glycerol
• Tip is irradiated with a beam of Xe0 or Cs+ which causes
sputtering of ions into the gas phase
• Good for polar, thermally labile species but sensitivity can be
3-6 orders of magnitude lower than ESI
15
Electrospray (ESI)
•Electrospray (nanoESI) •Pneumatically assisted electrospray

•up to 1 µl/min • flow rate range 5µl/min –
1mL/min
•Concentration dependant
• sensitivity poorer than nanoESI
•Most sensitive
• Concentration dependant
• More robust
16
ESI
In-line spraying - most modern

sources are orthogonal ie at right
angles to the entrance to the MS
• All API sources/interfaces

work because while the
source is at atmospheric
pressure the MS analyzer is
at high vacuum.
• A series of skimmers and
vacuum pumps is placed
between the source and the
MS to reduce pressure from
AP to HV
• Orthogonal spraying helps
reduce the gas load into the
17
MS
ESI – Remember!
• Droplets are highly charged during the spraying process and

Coulombic repulsion causes the spray to “explode” into
smaller drops as the solvent evaporates
• As the droplets become smaller the charged analyte ions will
be desorbed into the gas phase (IEM) or as the droplet
becomes completely dry (CRM) and then extracted into the
MS
• Orthogonal source designs now ie sprayer at right angles to
the entrance to the MS
18
APCI
19
APCI
• Advantages:
– Entire mobile phase and sample vaporized into the gas phase
(with heat), then ionized
– Accommodates high LC flow from (0.2 - 2 mL/min)
• Uses heat (400–500 °C) and nebulizer gas to vaporize
HPLC eluent and transfer sample into the gas phase for
APCI
– Temperature setting is not critical
– Sensitive
• Disadvantages:
– Thermally labile analytes may degrade when vaporized
– Low molecular weights only
20
– CI mass spectra only, little fragmentation typically
APPI
courtesy of Applied Biosystems

21
APPI
Advantages:
– Entire mobile phase and sample vaporized into the gas phase
(with heat), then ionized
• Uses dopant (Toluene/Acetone) to promote ionization
– Accommodates high LC flow from (0.2 - 2 mL/min)
• Uses heat (400–500 °C) & nebulizer gas to vaporize HPLC
eluent and transfer sample into the gas phase for
photoionization
– Detection limits are very good
• Improvements over ESI and APCI compound dependant
– Wider application range (low to medium polarity compounds)
Disadvantages:
– Thermally labile analytes may degrade when vaporized
– Low molecular weight compounds only (<1000) 22
Moving Belt
3 4
A
B 2 1
• LC eluent is mechanically transported from the column (A) to the

high vacuum ion source (B)
• Combination of differentially pumped regions (1,2,3) and heating (4)
removes solvent
• The dried sample is flash vapourized into the source (EI/CI) or
23
irradiated with Xe0 or Cs+ ions (FAB/LSIMS)
Moving Belt
• The polyimide belt contributes to the chemical noise of the
system
• Mobile phases containing high H2O concentrations are
difficult to handle
• Latent heat of vapourization of H2O is very high
(compared to organic solvents)
• Can bead on the belt (high surface tension) – destroys
chromatographic integrity
• Mechanically very complex - poor reliability

• One of the first LC/MS interfaces available (along with DLI)
24
Particle Beam
25
Particle Beam
• LC eluent introduced with He to generate an aerosol of solvent droplets

• Solvent evaporates and the resultant beam of dry particles, He and
solvent vapour enters a momentum separator which preferentially
removes low mwt species (He and solvent)
• Dry particles enter a conventional high vacuum EI/CI source
• 0.1 – 1mL/min flow rates accommodated
• Samples must be volatile and thermally labile and mwt<1000
26
Tandem Mass Spectrometry – MS/MS
• The combination of 2 or more stages of mass analysis in one experiment
• These stages are decoupled from one another in one of 2 ways:

– MS/MS in space, where parent ion selection, dissociation and
subsequent mass analysis are physically performed in different regions
of the MS eg BE/EB, QQQ, Tof-Tof etc
– Hybrid instruments, where 2 different types of mass analyzer are put
together eg QTof, QTrap etc
– MS/MS in time, where parent ion selection, dissociation and
subsequent mass analysis are performed in the same region of the MS
but are decoupled in time ie they are performed as a series of timed
events following one another eg 2D and 3D ion traps, FTICRMS
• The dissociation step is typically achieved using Collision Induced

Dissociation (CID) sometimes called Collisional Activation (CA)
27
Ion Stability
• 3 “types” of ions based on the mass spectrometric time frame:
– Unstable – dissociate quickly ie before leaving the source k >
106s-1 and are never observed
– Metastable – dissociate after leaving the source but before
detection 105s-1 < k < 106s-1 can be detected
– Stable – arrive intact at the detector k < 105s-1
• In order to perform MS/MS we must somehow destabilize the

“stable” ions and cause them to dissociate
• There are a number of ways to accomplish this but the most
common is Collision Induced Dissociation (CID)
28
Collision Induced Dissociation (CID)
• An ion/neutral species interaction wherein the projectile ion is

dissociated as a result of interaction with a target neutral species
(N2, Ar, He). This is brought about by conversion of part of the
translation energy of the ion into internal energy of the ion during
collision.
AB+ + Ncg AB+* A+ + B + Ncg

vibrationally and
electronically excited
• The internal energy (IE) of AB+* is composed of IE prior to collision

(usually low) and the amount Q, transferred during collision:
EAB+* = EAB+ + Q 29
CID
• The absolute upper limit for Q is defined by the “centre of mass” collision
energy, ECM)
ECM = ELAB
( m N
__________
mN+mAB )
Where: ELAB user set collision energy
mN is the mass of the target or collision gas
mAB is the mass of the ion
• ELAB can be in the 1-10keV range for BE/EB and Tof-Tof instruments – single or
double collisions, He employed with short collision cells
• ELAB can be in the 1-200eV range for QQQ, QIT, Q-Tof instruments employing
multiple collision events with Ar, N2 or He (for QIT) and longer collision cells
• Scattering can be an issue
• Very fast process – 10-15 s
• Must not ionize the collision gas in the collision event
30
CID
• For example:
– an ion of m/z100 with a collision energy of 50eV colliding with
He will gain a maximum of 50*(4/(100+4)) = 1.9eV.
– consequently, in the 1-200eV collision regime, we usually
employ N2 or Ar as the collision gas to maximize the energy
transfer
– the same ion with 5 keV, colliding with He could in principle

pick up 190eV of internal energy, and no doubt some do, but
very, very few are seen.
– consequently, in the keV collision regime, we usually employ
He as the collision gas to minimize ion scattering and prevent
excessive fragmentation
31
CID
• In high energy CID in a sector instrument, we expect to see only the products of
glancing collisions which do not knock the ions out of the beam, and because such
experiments are carried out under single-collision conditions (each precursor ion
undergoes a maximum of one collision), the total internal energy is relatively low,
say 1 – 15 eV.
• As a general rule, the bigger it is, the harder it is to break. A simple way of
understanding this is to keep in mind that the energy deposited in the ion may not
be localized, and in any case, rapidly distributes over the bonds of the ion. Bigger
ions Æ more bonds Æ less energy/bond, and less chance that a given bond will
have enough energy to break.
• A CID type process can also occur in the source of an API instrument because ions
are accelerated (focused) through a high pressure region before they enter the
mass analyzer – this can cause “unstable” ions to collide with neutral gas
molecules and dissociate in the source – called “in-source CID”
32
“In-source” Collision Induced Dissociation (CID)
mwt=451
396.128
100
Ions moving quickly through
“normal” cone voltage
high pressure region
165V
[M+H]+
%
352.143
164.071
452.198
397.145 474.184
0 m/z
100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500
100
452.175 [M+H]+
“low” cone voltage Ions moving slowly through

65V high pressure region
%
396.128
453.198
0 m/z 33
100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500
Why Tandem Mass Spectrometry?
• To separate the ionization step from the fragmentation step

– for example, in an EI full scan mass spectrum of an analyte that
fragments extensively in the source, it can be difficult to determine
which fragment ion is formed from which precursor – MS/MS allows
individual ions to be selected and their fragmentation behavior
studied in isolation from any other processes
• Direct mixture analysis
• Increase specificity, reduce “chemical noise” for quantitative
studies (SRM/MRM)
34
Tandem Mass Spectrometry
• In the simplest of these experiments, MS/MS, the 1st mass
analyzer is used to transmit only one ion observed (m2) in the
full scan mass spectrum into the dissociation region
• In the dissociation region (collision cell), ions are “excited”
energetically in a variety of fashions depending on the
instrument type. This leads to the dissociation of m2 into
fragment ions
• The 2nd mass analyzer is scanned to pass in turn the products
of the dissociation of m2 onto the detector
• This is the simplest of the MS/MS experiments and is called a
product ion scan
• There are many others: precursor ion scan, neutral loss scan,
selected reaction monitoring
35
Tandem MS - Product Ion Scan Schematically
Full scan MS of analyte

m2
Mass select m2 and dissociate

m3 MS/MS spectra of m2
Mass select another ion (m3) from the

MS/MS spectrum of m2 and dissociate
MS/MS/MS spectra or MS3
36
Magnetic Sector Instruments
• Ion dissociation in a Field Free Region (FFR):
– Metastable decomposition
– Collision Induced Dissociation (CID)
– Dissociation causes partitioning of ion kinetic energy and momentum
between the 2 particles
• Constant Linked scan with EB instruments:

– To look at dissociations occurring in the 1st FFR
– Must scan B/E together (linked)
– Not true tandem MS because B and E are not operated separately
– Poor precursor ion resolution
• Mass analyzed Ion Kinetic Energy Spectra (MIKES):

– To look at dissociations occurring in the 2nd FFR
– Only on BE instruments
37
– Poor fragment ion resolution
Reminder: Metastable Ion (from section 3 (EI) pg7)
Fragment ions
M+.
Metastable ion
• M+. and fragment ions~0.3Da

wide
• Metastable ion~3Da wide!
38
Reminder: Metastable Ion (from section 3 (EI) pg7
• It’s position at m/z 200.3 can be used to determine which ion is fragmenting to give
which product:
For the reaction, M1+ M 2 + + M3
apparent mass of the metastable, M* = M22
____
M1
• In this case, solving the equation: M1+ is m/z 255 and M2+ is m/z 226
• Therefore this metastable ion corresponds to the fragmentation of the M+. ion to
yield the fragment at m/z 226
• the other metastable ion at m/z 146.6 corresponds to m/z 226 fragmenting to give
182 39
Magnetic Sector Instruments
• To overcome the resolution issues other sector based

instruments have been built eg BEBE, EBEB etc
40
Triple Quadrupole – MS/MS in Space
• Basic construction
– QQQ configuration: Q1 and Q3 are mass filtering
with Q2 being the collision cell
– Ar or N2 collision gas employed for MS/MS
Q1 Q2 Q3
Ion source Detector
Product Ion Scan
Park Q1 to Allow Only Ions of a Single m/z ratio to pass into Q2
These ions collide with collision gas at a given CE and dissociate (CID)
Q3 is Scanned yielding the Full Scan Product Ion Spectra 41

QQQ – Full Scan Mass Spectrum
Q1 Q2 Q3
Transmits all ions Transmits all ions Scan
• Full scan mass spectrum of all source generated ions
Typical ESI Full Scan MS: Q3 scan, Q1 and Q2 Rf only mode
[M+H]+
little or no fragmentation
observed
42
QQQ – Product Ion Scan
Q1 Q2 Q3
Selected Ion CID Scan
• Full scan MS/MS spectrum of mass selected Parent Ion

• Used for structural elucidation
Typical ESI Full Scan MS: Typical ESI Product Ion Scan:
Q3 scan, Q1 and Q2 Rf only mode Q1 selects (M+H)+, CID occurs in Q2
[M+H]+ Q3 scanned
little or no fragmentation
observed
[M+H]+
43
Precursor Ion Scan
Q1 Q2 Q3
Scan CID Selected Ion
• Q3 is set to transmit only 1 ion characteristic of a specific

class of compounds eg M1 = m/z 79 (PO3)-
• Then as species are introduced into the source, Q1 is
scanned and only those compounds that fragment to
yield M1 will be detected
• Used for target compound class detection usually with
chromatography
44
Precursor Ion Scan
Q3 monitoring only m/z 79
Intensity
Intensity
Time
Time
• β-Casein Digest Full scan
• Complicated with many peptides • β-Casein Digest Precursor Ion Scan
• Detection of only Phosphorylated peptides
45
Courtesy of the MSCLS at the Univ. of Minn.
Neutral Loss Scan
Q1 Q2 Q3
Scan CID Scan at Q1-neutral mass
• Q1 and Q3 are both scanned however there is a mass

offset (Mn) between Q1 and Q3
• Then as species are introduced into the source, only those
compounds that loose the neutral mass, Mn, will be
detected
• Used for target compound class detection usually with
chromatography
46
Selected (Multiple) Reaction Monitoring –
SRM/MRM
Q1 Q2 Q3
Selected Ion CID Selected Fragment
• From a complex mixture, Q1 is set to pass only the parent ion from a
specific analyte, M1, and Q3 monitors only 1 of the unique fragment
ions formed from M1
• Ideally this allows the unambiguous detection of only 1 (or more)
analyte in a very complex mixture
• Used for target compound quantitation usually with chromatography
• The most sensitive and specific of all MS techniques for quantitation
47
3D Quadrupole Ion Trap – MS/MS in Time
Remember:
z He buffer gas necessary for efficient trapping of ions directed

into the ion trap
z He present at all times therefore no delay between MS and
MS/MS experiments Ring
Endcap
+
+ + +
+ +
+
+ +
+
+
+ detector
Ions gated into trap
Endcap
QIT (ion isolation)
• An additional Rf voltage is applied to the end caps.

• All but ions with one mass become non-stable and are
ejected from trap
• Resonance excitation of the trapped ion with additional Rf
voltage → oscillations of trapped ion increases
• The trapped ion collides with helium atoms and become
internally excited
• Collision Induced Dissociation (CID)
• New mass scan → MS/MS or (MS)3 spectrum
• In analogy: (MS)3, (MS)4, ….
49
QIT: MS/MS Scan
• 1: Clear Trap
• 2: Accumulation Time
• 3: Isolation Delay
** **
• 4: Isolation begin
• 5: Fragmentation
delay
• 6: Fragmentation
** *
begin*
* * *
• 7: Scan delay
• 8: Mass Analysis**
50
Courtesy of Agilent
3D - QIT
• Can only perform product ion scans – no precursor, neutral

loss or SRM functionality
• Most often used in qualitative analysis ie structural
elucidation
• In scanning mode (MS, MS/MS), much more sensitive than a
Q or QQQ
• MS/MSn
51
QQQ with Linear Ion Trap - QTrap
Dipolar Aux AC
Skimmer
N2 CAD Gas
Q0 Q1 Q2 Q3
IQ1 Exit
IQ2 IQ3
LINAC linear ion trap

3x10-5 Torr
52
Q-Trap
• All the functionality of a QQQ
– Product ion Scan
– Precursor Ion Scan
– Neutral Loss Scan
– SRM
• with the additional advantage that Q3 can be switched (software) into

trapping mode operation
– Very high scanning sensitivity compared to Q operated with
AC (Rf) and DC
– Linear trap also has some advantages over 3D QIT
• Can perform quantitation and structure elucidation (MS/MS3) sequentially

in a chromatographic time frame 53
QQQ with Linear Ion Trap - QTrap
Dipolar Aux AC
Skimmer N2 CAD Gas
Q0 Q1 Q2 Q3
IQ1 Exit
IQ2 IQ3
LINAC linear ion

trap
3x10-5 Torr
MS3 - Implementation
• Precursor ion selection in Q1.
• Fragmentation in Q2.
• Trap products in Q3.
• RF/DC isolation in Q3.
• Single frequency excitation in Q3.
• Mass scan. 54
Complementary MS/MS Approaches:
•Tandem in Space: Triple Quads
•Poor scanning sensitivity
•Great for quantitation (SRM/MRM)
•Very selective scans
•No low mass cut-off
•Tandem-in-Time: 3D Ion Traps

•Very sensitive scanning
•Only product ion scans - MSn
•Only scanning
•Low mass cut-off!
•3D vs 2D (linear) Traps

•Linear traps have higher charge capacity
•No low mass cut-off
55
•Linear traps restricted to MS3
Hybrid Quadrupole Time of Flight - QTof
• a QQQ where Q3 has been replaced by a reflectron TOF MS
• TOF used to acquire (accumulate) both MS and MS/MS spectra
• TOF accepts ions in a “pulsed” mode from the continuous ion beam
passed to the pusher by the optics
56
Courtesy of Waters
Q-Tof
• Advantages:
• High resolution (104) and accurate mass
• good scan sensitivity
• MS and Product Ion MS/MS
• Disadvantages:
• historically, TOF instruments suffer from a “poor”
dynamic range compared to Q instruments – this is
changing
• no Precursor Ion, Neutral Loss or SRM/MRM capability
57
+ve ESI - Peptide MS
Glu-Gly-Val-Asn-Asp-Asn-Glu-Glu-Gly-Phe-Phe-Ser-Ala-Arg
E-G-V-N-D-N-E-E-G-F-F-S-A-R
mwt = 1569.67
100
[M+2H] 2+ 785.82
786.32
797.31
%
804.80
805.29
815.79 [M+H]+
411.26 816.29
203.05
219.02 355.07 816.79
776.82
149.02 834.77 1570.67
0 m/z
200 400 600 800 1000 1200 1400 1600
58
+ve ESI - Peptide MS/MS
175.13
100
MS/MS of [M+H]+ CE = 85eV Ar
1570.80
684.37 1571.76
1570.59
684.41
1056.54 1571.87
%
1056.60 1570.51
1571.90
685.40 1057.56 1572.81
813.47 1056.36
316.18
333.20 497.24 684.29 813.38 814.48 1039.53 1057.63 1570.40 1572.89
942.44 1573.89
0 m/z
200 400 600 800 1000 1200 1400 1600
684.38
100
787.86
787.83 788.35 MS/MS of [M+2H]2+ CE = 35eV Ar
813.45
187.08 333.21
480.29
1056.53
%
240.15 685.39 1056.57

814.42 942.44 1285.65
1171.57
685.43 1286.60
337.18 497.22 943.51 1172.56
627.36 924.44 1286.71
382.20 498.21 740.30 1058.54 1287.59
815.46
0 m/z
200 400 600 800 1000 1200 1400 1600
Doubly charged peptide ions yield more sequence ions than does the singly
59
charged counterpart!
How Do Peptides Fragment?
x3 y3 z3 x2 y2 z2 x1 y1 z1 H+
R1 O R2 O R3 O R4
H2N C C N C C N C C N C COOH
H H H H H H H
a1 b1 c1 a2 b2 c2 a3 b3 c3
– If this charge is retained on the N terminal fragment, the

ion is classed as either a, b or c
– If the charge is retained on the C terminal, the ion type
is either x, y or z
– A subscript indicates the number of residues in the
fragment
60
+ve ESI - Peptide MS/MS Sequencing
y13 y12 y11 y10 y9 y8 y7 y6 y5 y4 y3 y2 y1
E--G--V--N--D--N--E--E--G--F--F--S--A--R
b1 b2 b3 b4 b5 b6 b7 b8 b9 b10 b11 b12 b13
MS/MS of [M+2H]2+ CE = 35eV Ar

684.38
100
y6 787.86
787.83 788.35
y 7
b2 y3
333.21 y4 813.45
187.08 480.29 y9
y8
1056.53 y11
%
240.15 685.39 1056.57 y101285.65

814.42 942.44
1171.57
685.43 1286.60
337.18 497.22 y5 943.51 1172.56
627.36 924.44 1286.71
382.20 498.21 740.30 1058.54 1287.59
815.46
0 m/z
200 400 600 800 1000 1200 1400 1600
61
• Low Energy CID (10eV to 100eV)
– collision induced dissociation in a QQQ, IT or QTof
– a peptide carrying a positive charge fragments mainly along its
backbone, generating predominantly a, b and y ions
– In addition, ions which have lost ammonia (-17 Da) denoted a*, b* and
y* and water (-18 Da) denoted a°, b° and y° are often observed
– Satellite ions from side chain cleavage are not observed.
• High Energy CID (keV)

– collision induced dissociation in a Tof-Tof or a magnetic deflection
instrument
– All of the ion series described above are observed in high energy
collision spectra. Relative abundances are composition dependent.
– Unlike low energy CID, ions do not readily lose ammonia or water.
– In addition, side chain cleavages can be observed, so called d, v and w
cleavages
62
Others?
• Of course:
• Orbitrap (linear trap – new type of 3D IT)
• Tof-Tof
• Linear trap-FTICR
• Trap-Tof etc etc
• Ion mobility coupled to other types of mass
spectrometer eg QTof where the collision cell has
been changed to allow IMS to be performed as well as
conventional MS/MS expts
63
Quantitation in GC-MS and LC-MS
• Every ionization technique exhibits a compound-dependent
response – that is, the same amount injected of different
analytes will give a different MS response
• In general, every aspect of the MS experiment can influence
the response from the analyte
• Ionization method
• Type of MS
• Chromatographic method
• Detector system
• Therefore a careful calibration of the instruments response

versus the sample concentration is a pre-requisite for
reliable quantitation
• Most MS quantitation methods employ chromatography 1
Important Definitions
• Limit of detection (LOD)
– How small an amount can you see? Usually this is defined as the
amount that will give a S/N of about 3.
• Limit of quantitation (LOQ)
– How small an amount can be quantitatively measured? Usually
significantly higher than the LOD.
• Sensitivity
– What’s the response per unit of analyte?
• Specificity
– Is the instrument response due only to the analyte? How well does
our analysis cope with interferences?
• Linearity
– Over what range is the response linear?
2
Important Definitions
• Calibration standards
– Prepared samples of known concentration for which corresponding
responses can be obtained
– Peak Area is most often employed as the response for mass
spectrometric assays however Peak Heights can also be used but
introduce a source of error
• Standard or Calibration Curve
– A series of standards covering the analyte concentration range
expected in the samples
– Mathematical function or equation describing the relationship between
instrument/ionization method/detector response and concentration
• Principle
– Mass spectrometric quantification can be done ONLY by reference to
standards (External or Internal)
3
Precision and Accuracy
A B
. ..
. .... .......
..
C . . .. D
......
.
. .
. .
. .
A: Accurate and Imprecise Precision (reproducibility):
B: Accurate and Precise GC-MS < 1%
C: Inaccurate and Imprecise HPLC-MS (electrospray):
D: Inaccurate and Precise Quadrupole ~ 1-2 % 4
Ion trap ~ 8 %
• Calibration
– In general, the response of a mass spectrometer to a specific analyte
will vary significantly with time
– Some of this variability can be directly attributable to parameters
directly related to the ionization process and use of the MS as the
detector however there are other sources of error:
• injection reproducibility when chromatography is employed
• Sample handling and preparation
• Why?
– over time, MS performance will decrease as a result of detector aging
and source/ion optics becoming contaminated
– sample components causing ion suppression or isobaric interferences
– this causes systematic variation in response/unit of analyte injected
5
• Solution:
– Quantitation calibration must be performed in real time to overcome
these issues
– 3 types of Calibration Methodologies:
• External Standard Calibration
• Standard Addition
• Internal Standard Calibration
6
• External Standard:
– Construct a calibration curve by injecting a series of analyte standards
and plotting response against concentration
– The samples are then analyzed and their response is compared to the
standard analyte response to derive their concentration
• However:
– Number of ions produced by a given analyte at a given concentration
varies with time therefore calibration curve less stable
– How closely does the standard matrix resemble the sample matrix?
– Did the MS response change between the analysis of the standards
and samples?
– These issues as well as some preparation errors can be overcome by
using an internal standard method
7
• Standard Addition:
– The unknown sample is divided in 2 portions and a known amount of
the analyte is spiked into one portion
– Samples measured both before and after addition
– The spiked sample shows a larger response and the difference in
response between the spiked and unspiked is due to the spike and
provides a calibration point to determine the amount of analyte in the
original sample
– Calculation of analyte concentration requires analysis of multiple
samples of each analyte ie with and without standard added
• However:
– A linear response is assumed when a 2 point determination is made,
that is, no calibration curve
8
• Internal standards (ISTD):
– A known amount of a reference compound (the internal standard) is
added to every sample
– If it is added before sample workup/extraction and if it has similar
chemical properties to the analyte then it can be used to compensate
for:
• differences in recovery during sample preparation (extraction)
• Ion suppression by residual matrix components
• Instrumental variability (injection volume etc)
– Chemically as similar as possible but not the analyte itself (obviously)
– no co-elution with matrix components
– However, we must have analyte free (blank) sample matrix to prepare
calibration curve and QC’s
– Gives improved accuracy and precision because an internal
reference is in every sample and is the method of choice for MS
quantitation 9
• Internal standard selection:

– isotopically labeled species is best
(13C/15N>2H)>homologue>>analogue
– Could use an isomer of the analyte but if the MS/MS behavior is similar to
the analyte then it must be chromatographically resolved from the analyte
– 13C or 15N labeled standards preferred
– Usually deuterated (2H ) standards are used as they are more available than
13C or 15N
– The label must not be able to be exchanged during the analysis ie 1H/2H
exchange – we need stably labeled ISTD’s
– several isotopes to avoid overlap with isotopic peaks of unlabelled analyte
>3 labels is optimum
– In multi-component assays, the ideal situation is to employ an internal
standard for each analyte
10
Schematic Representation of a Typical
Quantitative MS Procedure
1 CRUDE 2 ANALYTICAL 3 RATIO OF RESPONSES

SAMPLE
EXTRACT SAMPLE [ANALYTE(S) TO ISTD(S)]
4
1a. Add internal standard(s) QUANTITY OF

1b. Homogenize ANALYTE
1c. Extract analytes and ISTD’s IN SAMPLE
2a. Purify (chromatography, further extraction etc)

2b. Concentrate if necessary
2c. Derivatize if necessary
3. Mass Spectrometric measurement

employing SIM or SRM with GC/MS(MS)
or LC/MS(MS)
4. Comparison with calibration curve 11

Quantitation: Sample Introdution
• Direct introduction:
• Fast and simple
• Dirty samples will require extensive cleanup to minimize suppression
and interference (keep in mind that choice of ionization methods differ in
their susceptibility to these problems)
• Chromatography/MS:
• Not so simple to develop/find a method and then you need to solve any
compatibility issues eg involatile buffers, ion pair reagents
• Takes longer
• Many problematic impurities can be separated from the analyte, eg salts
which interfere with ESI will elute in the void volume of a reverse phase
column - less sample cleanup may be required
• Keep in mind that LC columns are not infinitely tolerant of “junk”, and
are not cheap!
• We may want to compare to say, a HPLC run using UV detection
• Adds the specificity of retention time to the analysis, essential if we are
using an internal standard of the same molecular weight 12
Which Ionization Technique to Chose?
• Electrospray (ESI)
– little or no heat applied excellent for thermally labile molecules
– polar (ionic) to relatively non-polar molecules
– LC flow: <1 to ~1000µL/min
– Most susceptable to matrix effects ie SUPPRESSION
• APCI & APPI

– Heat applied to vapourize sample and mobile phase therefore not for
thermally labile molecules!
– medium to low polararity analytes
– LC flow: 100 – 2000µL/min
– Less prone to matrix effects
• Which technique gives the best response (sensitivity)

for your analyte?
13
Methomyl
ESI APCI APPI

347.1
185.0
163.1
Max: 206617
90000 Max: 3663 Max: 95891
90000 90000
80000
80000 80000
70000
70000 70000
60000
[M+H]+= 163 60000 [M+H]+= 163 60000 [M+H]+= 163
50000
50000 50000
40000
348.1
40000 40000
165.1
30000
30000 30000
163.1
20000
106.1
20000 20000
164.1
186.1
163.1
10000
10000 10000
0
0 0
200 400 600 800 m/z 200 400 600 800 m/z 200 400 600 800 m/z
courtesy of Agilent
14
• Full scan:
– Extracted ion chromatogram
– Specificity based on mass alone eg (M+H)+
– Poor sensitivity compared to SIM and SRM
• Selected Ion Monitoring (SIM)
– Specificity based on mass alone
– More sensitive than full scan as we are only monitoring a few ions
rather than the full mass range
• Optimum:
– Selected reaction monitoring (SRM, MRM) in MS/MS with QQQ
– Specificity based on mass and unique fragmentation
– MS/MS with ion trap/QTof – no SRM mode
– Significant reduction of chemical noise (less sample preparation)
– Best S/N (sensitivity) of all 15
Full Scan vs SIM vs SRM
Same sample injected in different

MS modes - increased specificity
and detectability!
16
Calibration Curve with Internal Std
Peak Area Peak area Ratio = area of analyte response
Ratio area of ISTD response
QC3
.
.
.
QC2
. Stds, samples,QC’s and
. blanks ran in same “batch”
QC1 .
.
. Analyte amount
• The “batch”:
– Blanks, std curve (S1-S8), 2QC samples (QC1 and QC2), ½ of
the unknowns, 2QC samples (QC1 and QC3), ½ of the
unknowns, 2QC samples (QC2 and QC3)
17
Calibration Curve with Internal Standard
• Blanks are very important:
– matrix blank: where matrix containing none of the analytes is

extracted using the same sample preparation scheme
– Internal standard blank: to assess whether the ISTD causes a
response in the analyte channel
– Solvent blank: does the solvent used to re-dissolve the extracted
sample interfere
– in many cases interference determines limit of quantitation (LOQ) and
not the absolute detectability (LOD)
• QC’s
– Samples of known concentration extracted along with unknowns to
assess method performance
18
Calibration Curve – General form
Absolute Signal Peak area Ratio = area of analyte response

Intensity area of ISTD response
or Peak
Area ratio .
. saturation
.
.
chemical bkgd . Linear range
or memory .
.
. adsorption
Noise level
concentration or amount
19
Linear Dynamic Range
• Some mass spectrometric methods are more linear than

others:
– EI has the greatest linearity, say 5 to 7 orders of magnitude
– ESI is probably the poorest exhibiting ~ 3 orders of magnitude
– APCI/APPI is a little better with ~ 4 orders of magnitude
– Trapping instruments tend to have lower dynamic range than Q type
instruments because they fill up ie space charging
– TDC with MCP type detectors used in Tof instruments are usually
limited to ~3 orders of magnitude, whereas electron multipliers are
much better ~ 6
– Linear or Quadratic regression is used to “fit” the calibration curve
20
Ion Suppression
• Suppression:
– Competition and interference with analyte ionization resulting in
decreased number of [M+H]+ or [M-H]- ions
• Caused by salts that form adducts and clusters with analyte:
– Strong bases in positive mode eg Triethylamine (TEA)
– Acids in negative mode eg Trifluoracetic acid (TFA)
– Non-volatile buffers (phosphate) and ion pair reagents
– Non-covalent dimers [2M+H]+, trimers....
– Metal ion complexes, e.g. [2M+Cu]+, Cu+ eg from LC equipment
– Other conatminants in the system eg PEG, platicizers, residual matrix
species
– Coeluting analytes with different proton affinities that is, analytes that
compete for protons
21
Ion Suppression: Solutions
• Dilute sample (1:10+) before or after sample preparation

• Select ISTD to coelute with analyte:
- to achieve similar matrix effect for analyte and ISTD (compensation)
- ideally the stable isotopically labeled analyte (2H, 13C, 15N)
- force chemical analogue into coelution with analyte
- individual ISTD for each analyte, if analytes are chromatographically
resolved
• Modify LC mobile phase composition, change stationary phase or
perform a more extensive sample clean-up
• In general, the more complex the sample, the more likely it is that
chromatography will improve your quantitative method
• Try APCI (or APPI) instead of ESI (if analyte is thermally stable)
22
Hints on Method Development
• Some form of sample preparation is critically important for rugged
and robust quantitative measurements:
– Solvent (liquid/liquid) extraction
– Solid phase extraction
• Prepare (external) calibration standards in same matrix as samples

• Internal Standard should match analyte structure as closely as possible
- goal is coelution of ISTD and analyte
• Check mass spectrometric interference with coeluting compounds
– interferences from analyte isotope peaks (eg Cl, Br, S)
– metabolites and their fragmentation to the analyte
• perform as much chromatography as is necessary
23
Ion Focusing
• Confine ions from dispersive environments and focus them spatially,
temporally and/or energetically at a point in space or on a relevant detector
to improve MS response
• We can focus ions because their energy and/or the momentum is
changeable – remember charged particles in an electric field
• Ion optics need to be in a vacuum (greatest effect)
• Accomplished using electrostatic lenses (plates, orifices, grids) or multipoles
placed in or close to the ion beam to cause ions to be deflected/focused
• Lens stacks for fast moving ions
– While a charged particle is in an electric field force acts upon it. The faster the
particle the smaller the accumulated impulse (rate of change of momentum =
mΔv)
– These plates can be stacked with as many as 30 sets to effect efficient focusing
– very complicated
• Simple lenses for slow moving ions – Einzel lens 1
Electrostatic lenses
• Basic types
– Immersion/aperture lenses – lens stacks and grids (shown below)
– Unipotential lenses – Einzel lens
Ion Beam
• All lenses are convergent

• Focal point is independent of m/z
2
Einzel lens
• used in Tof’s, quads, ion entrance optics

• Focuses ions without changing the kinetic energy
3
Ion Focusing
• quadrupoles (also hexapoles and Without collisional
octopoles) in Rf only mode ie no DC cooling
voltage (a=0) act as wide band pass
filters for ions - very efficient especially
at higher pressures
• Collisional cooling of ions traveling
slowly through Rf only multipoles with
some collision gas present reduces
With collisional
the axial motion of ions and therefore cooling
increases the mass resolution (used in
QTof instruments)
4
Ion Detection
• The Faraday Cup:

• The ion beam impacts the FC and deposit their charge – the
resulting current flowing away from the FC results in a voltage
that can be measured
• Used rarely except in isotope ratio MS ie very accurate but not
very sensitive
5
Discrete Dynode Electron Multiplier
• When an energetic particle (a positive or negative ion) impinges on the
surface of a metal or semiconductor a number of 20 electrons are
emitted from the surface
• The ease of such emission is determined by the electron work function
(we) of the respective material eg BeCu alloy oxide (we = 2.4eV) and
the velocity of the impacting particle
• The higher the velocity of the impacting particle and the lower the we
the larger the number of 20 electrons formed:
– larger, slow moving ions will yield fewer 20 electrons than small, fast
moving ions which leads to high mass discrimination
– Can be solved by employing a post-acceleration conversion dynode
(PACD)
6
Discrete Dynode Electron Multiplier - no PACD
• If an electrode opposite to the location of 10 emission is held at a more

positive potential, then all emitted electrons will be accelerated towards
and hit the surface where they in turn cause the release of even more
electrons
• With 12 to 18 discrete dynode stages held at about 100V more positive
7
potential allows the ion signal to be detected by a sensitive preamplifier
Discrete Dynode Electron Multiplier – no PACD
• Normally have a gain of 106 – 107 (when new)

• Have a lifetime or around 1 – 2 years before having to be
replaced
• Must be kept at high vacuum as the emission layers can be
harmed by oxygen
• over time and with use the first dynode surface becomes
damaged and is therefore less efficient at releasing electrons ie
gain is reduced
• This can be compensated for to some extent by increasing the
voltage difference between the discrete dynodes however
eventually the multiplier must be replaced
8
Electron Multiplier with Post-Acceleration
Conversion Dynode
(ion beam)
20 electrons Dynodes Electron collector
Conversion Further
Dynode Amplification
and recording
Potential gradient
• All ions are accelerated to high velocity by the Post-Acceleration Conversion Dynode
• All ions release ~ the same number of 20 electrons therefore high mass discrimination
greatly reduced! 9
Channeltron Multiplier – continnuous dynode
• The inner surface is composed of a layer of silicon dioxide over a conductive layer
of lead oxide
• This surface has sufficiently high resistance to withstand the ~1.5 to 2.5kV placed
across the multiplier
• The high voltage drops continuously from the entrance to the exit of the tube
• Gain of around 105 to 106
• Ages in a similar fashion to a discrete electron multiplier
• An array of linear channeltron multipliers is called a microchannel plate (MCP) –10
each multiplier is of the order of a few micrometers in size
Microchannel plate (MCP)
Ions
• Channels are inclined by some degrees from the perpendicular so that ions
strike the inner surface and cause 20 electron emission
• Gain is only ~ 103 to 104 so sometimes 2 MCP’s are sandwiched together
so that the small offset angle of the channels oppose each other to form
what is called a chevron plate 11
Reflectron Tof Head
Tof Pusher
MCP
12
Courtesy of Waters/Micromass
Photomultiplier with Conversion Dynode
• The 20 electrons from the conversion dynode strike a phosphor which

emits photons
• The photomultiplier detector has a much longer lifetime than an electron
multiplier as it is a sealed device (under vacuum)
13
• 107 gain
Photomultiplier with Conversion Dynode
• Advantages:
– Long lifetime and gain does not decrease with time (compared with
an electron multiplier)
• Disadvantages:
– dark current – when an intense signal ie many electrons strike the
phosphor it can take some time for the phosphor to stop releasing
photons even when no electrons are hitting it
– Causes a signal to be recorded when there is no signal
14
Ionization Technique Summary
Ionization Polarity Thermally Mwt Examples
EI Low stable <1,000 Non-ionic organics
CI Low stable <1,000 Non-ionic organics
FI Low to med stable <1,000 Non-ionic organics
FD Low to med labile <2,000 Non-ionic organics,

peptides, organometallics,
carbohydrates
FAB/LSIMS Med to high labile <20,000 Polar/ionic organics,

peptides, biomolecules,
organometallics
MALDI Med to high labile <200,000 Peptides, proteins, RNA,

DNA, polymers
ESI Med to high labile <100,000 Polar/ionic organics,

peptides, proteins,
biomolecules, polymers,
organometallics
APCI/APPI Low to med stable <1,000 Non-ionic organics
1
MS Experiment Summary
Experiment Ionization Instrument Type Comment
Batch All All DIP (EI, CI, FAB/LSIMS
Introduction and MALDI)
Infusion (ESI and CF-FAB)
Low resn all Q, QQQ, EB/BE, linear Mass spectrum (some
and 3D IT, linear Tof database searching
capability for EI)
High resn EI, FAB/LSIMS, EB/BE, reflectron Tof Accurate mass yields
and FTICRMS elemental composition
MALDI, ESI
MS/MS all QQQ, linear and 3D IT, Structural elucidation and
QTof, and FTICRMS quantitation (SRM/MRM)
(other hybrids)
GC/MS EI, CI, FI EB/BE, Q, QQQ, 3D-IT, Complex mixture analysis
Tof of semi-volatile species
LC/MS ESI, APCI, Q, QQQ, linear and 3D Complex mixture analysis

APPI IT, Tof, QTof, FTICRMS of more polar species
2
Mass Analyzers Summary
Analyzer Ion Ionization Mass Resolution
Detection Modes Range
Sector continuous EI, CI, ~15,000 Variable up
(BE/EB) FAB/LSIMS to 100,000
Quadrupole continuous EI, CI, ESI, APCI, 4,000 Normally 3,000
(Q and QQQ) APPI but 10,000
possible
Ion Trap pulsed EI, CI, ESI, APCI, 4,000 ~3,000
(linear and 3D) APPI, AP-MALDI
FTICRMS pulsed EI, CI, ESI, APCI, 106 105 to 106

APPI, MALDI
TOF pulsed all Unlimited (L) ~1,000 (L)

10,000 (R) Up to 20,000 (R)
L = Linear Tof and R = Reflectron Tof

3

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Chem 425- Mass Spectrometry

Tuesday and Thursday: 10am – 11:20am, C2-361

Course TA: Jonathan Martens

• 4. Chemical Ionization (CI and DCI)

• 7. Laser Desorption Ionization and Matrix Assisted Laser Desorption

• 8. Inductively Coupled Plasma (ICP)

• 9. Atmospheric Pressure Ionization (API)

• 11. Hyphenation in Mass Spectrometry

• 13. Ion detection and Focusing

Sun Mon Tues Wed Thurs Fri Sat

Sun Mon Tues Wed Thurs Fri Sat

Sun Mon Tues Wed Thurs Fri Sat

Sun Mon Tues Wed Thurs Fri Sat

Jurgen H. Gross - Springer 2004 – QD96M3G76

• Mass Spectrometry: A Foundation Course

K. Downard – Royal Society of Chemistry 2004 – QD96M3D69X2004

• Chemical Ionization Mass Spectrometry – 2nd edition

Alex G. Harrison – CRC Press 1992 – QD96M3H371992

• Interpretation of Mass Spectra

Fred W. McLafferty – University Science Books

• International Journal of Mass Spectrometry

• Journal of the American Society For Mass Spectrometry

• Mass Spectrometry Reviews

• Journal of Mass Spectrometry

• Rapid Communications in Mass Spectrometry

Gas +ve and -ve ions

Mass separation using

• 1934: J. Mattauch and R. Herzog (Nobel prize)

• 1936: Secondary Ion Mass Spectrometry introduced

• 1940: Westinghouse Electric begins development of a portable MS for

• 1943: CEC installs 1st commercial MS at the Atlantic Refining Company

• 1945: CEC introduces 1st analogue computer for data analysis

• 1948: ICR MS “Omegatron“ is developed

• 1952: 1st A/D converter for data aquisition (CEC)

• 1953: W. Paul → Quadrupole mass spectrometer and ion trap detectors

• 1954: 1st high resolution MS developed at Imperial Chemical Industries

• 1956: PA determinations made by MS, McClafferty rearrangment

• 1958: CEC introduces the 1st digitizer. Bendix TOFMS introduced

• 1959: peptides and oligonucleotides sequeunced at MIT

• 1960: Bendix introduces 1st direct insertion probe

• 1966: Tandem MS for ion-molecule studies is developed

• 1968: Electrospray Ionization introduced at Northwestern U for studying

• 1971: relectron time-of-flight develpoed in Lenningrad

• 1975: mass spectrometers are placed on the Viking Mars missions

• 1979: Ion evaporation model developed (ESI)

• 1980: ICPMS is developed at Iowa State U. 1st commercial QQQs introduced.

• 1981: FAB introduced.

• 1986: LC interfaced to MS with pneumaticaly assisted ESI.

• 1988: Karas and Hillenkamp – MALDI

• 1994: Micro- and nano- ESI are introduced

• In the past 10 years a wide variety of hybrid instruments have been

Sample Introduction Ion source * Mass Analyzer(s) * Detector

Separates ions based Indirect

Gas or condensed Some form of Ions

Integrated with some degree of computer control 1

• All Ion sources produce either

• Odd electron ions eg M+. (EI, charge exchange CI)

• Computer employed for instrument control, data acquisition

• Fragment ions: [F]+ formed in the source by direct bond

• Metastable ions, m* are ions that fragment outside the ion

M+. and fragment ions~0.3Da wide

• Magnetic field causes e- to spiral

• We need to understand the processes involved if we hope