Mass Spectrometry

Dr Nusrat Subhan
 History of MS Applications
1899 Early Mass Spectrometry;
1956 Identifying Organic Compounds with MS;
1964 GC/MS
1966 Peptide Sequencing
1990 Protein Structure
1991 Non-Covalent Interactions with ESI
1992 Low Level Peptide Analysis
1993 Oligonucleotide Sequencing
1993 Protein Mass Mapping/Fingerprinting
1996 MS of a Virus
1999 Desorption/Ionization on Silicon
1999 Isotope-Coded Affinity Tags
 MS Celebrities
Ion Chemistry
Francis William Aston
(1877 - 1945)
Cambridge University, Great
Britain; Nobel Prize in Chemistry
1922
ESI of Biomolecules Fragmentation Mechanisms
1stMS John B. Fenn (1917) Fred W. McLafferty
Joseph John Thomson (1856 - Ion Trap Technique Virginia Commonwealth (1923)
1940) Cambridge University, Wolfgang Paul University, Richmond, Virginia Cornell University
Great Britain; Nobel Prize in (1913 - 1993) University of
Ithaca, New York
Physics 1906 Bonn, Germany; Nobel Prize
in Physics 1989

Peptide Sequencing using MS

Mechanisms and Applications Mechanism of MALDI & ESI
Klaus Biemann (1926)
MALDI R. Graham Cooks (1941) Michael Karas (1952)
MIT, Cambridge, Massachusetts
Franz Hillenkamp (1936) Department of Chemistry, Purdue University of Frankfurt,
University of Münster, Germany University Germany
West Lafayette, Indiana
 Introduction
• An analytical technique which is used for the
determination of the molecule mass and explanation
of the chemical structures of molecules, such as
peptides and other chemical compounds. (12X5)+(12X1)= 72

• It is most accurate method amongst all other

• Signal will appear in the form of
spectroscopic technique.
mass to charge ratio (m/e or
• Of all the organic spectroscopic techniques, it is used m/z)
by more divergent fields –molecular biology, • Generally, charge will be +1 in
semiconductors, geology, archaeology than any most of the cases, rarely it will
other. come +2, that means 72/+1= 72
 Bombarded e-
Principle e-
e-
e-
e-
• A beam of light will be bombarded in the
analyte compound, and it will lead to removal e-
M
of 1e- from analyte
M 2e-
e-
• Due to the removal of the e-, molecule will be +ꙩ
M - e- M + 1e-
positively charged and known as molecular Fragmented Fragmented
+ ꙩ ꙩ +
ions m1 + m2 m1 + m2

• Molecular ions will be fragmented and mass spectroscopy

Stable ions
only positive charged will be detected
M+ , m1+, m2+ can be detected
 Molecular ion (M+)
m/z = 16
Theory H H
- -
H CH + e H CH + 2e
• The impact of a stream of high +ꙩ
H C5H12 H
energy electrons causes the Neopentane
molecule to lose an electron Molecular ion Fragment ion

forming a radical ion.

+ + + +
• A species with a positive charge and CH 3 C2H5 C3H7 C4H9
ꙩ ꙩ ꙩ
one unpaired electron
C4H9 C3H7 C2H5 CH ꙩ 3

Radical ion
 H
Theory
H C H
• Molecular ion (parent ion):
H
• The molecular cation corresponding to the

H H
mass of the original molecule

• The molecular ion is usually the highest

mass in the spectrum H C C H
• Some exceptions w/specific isotopes

• Some molecular ion peaks are absent.

H H
• Mass spectrum of
ethanol (MW = 46)
M+

• Known molecule mass of ethanol is 46

(12X2+ 16X1 +1X6) =46
• A fragment generated by the loss of the -CH3
(46 – 15)= 31
• A fragment generated by the loss of the -OH
(46 – 17)= 29
Fragmentation
of Ethane
• The impact of the stream of high
energy electrons can also break
the molecule or the radical cation
into fragments.
 Theory
• Only ions are detected.

• Radicals are “invisible” in MS.

• The amount of deflection observed

depends on the mass to charge ratio
(m/e).

• Most cations formed have a charge

of +1 so the amount of deflection
observed is usually dependent on the
mass of the ion.
 Sample injection

m/z ∞ r2 Vaporization
m2 > m1
Ionization
r1
r2 Fragmentation

m/z ∞ 1/Deflection Acceleration

1) 16O2 + , 2) 16O 18O + Deflection

Mass separation
X
Neutral molecule
Detection
What’s in a Mass Spectrum?
• The resulting mass spectrum is a graph
of the mass of each cation vs. its relative
M+
abundance.

• The peaks are assigned an abundance as

a percentage of the base peak.
• the most intense peak in the spectrum

• The base peak is not necessarily the

same as the parent ion peak.
Relative abundance?

+
M 250
9000 molecules

F1+ F2 + F3+ F1+ F5+ M+ Mass spectrum

Mass 50 100 150 200 225 250

No 1500 500 1000 2000 3000 1000

Fragment No
𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝑎𝑏𝑎𝑛𝑑𝑎𝑛𝑐𝑒= Maximum X 100 1500 X100
RA=
3000
 Important information • The base peak in a mass spectrum is
• The heaviest ion in the mass spectrum (the one the most intense (tallest) peak
with the highest m/z value) is most likely the attributable to the ion with the
molecular ion. largest relative abundance (relative

• Or The molecular ion peak is the peak in a mass intensity; peak height along the y-

spectrum that represents the molecular ion axis).

(symbol: M peak).

• The molecular ion peak is the peak with the • Contrary to popular belief,
largest mass-to-charge ratio after excluding any molecular ions are not always
peaks caused by the presence of heavier base peaks, and base peaks are
isotopes. not always molecular ions.
 Important features of the Molecular/parent ion peak

(a) The molecular ion peak in aromatic compounds is relatively much intense due to
the presence of p-electrons.

(b) Conjugated olefins show more intense molecular ion peak as compared to the
corresponding non-conjugated olefins with the same number of unsaturation.
Conjugated olefins are more stable than the corresponding non-conjugated olefins.

(c) Unsaturated compounds give more intense peak as compared to the saturated or
the cyclic molecule.
 Important features of the Molecular/parent ion peak
(d) The relative abundance of the saturated hydrocarbon is more than the corresponding
branched chain compound with the same number of carbon atoms. For example, the molecular
ion peak for n-pentane is more intense than that of neopentane.

(e) The substituent groups like –OH, -OR, -NH2 etc. which lower the ionization potential
increase the relative abundance in case of aromatic compounds. Also the groups like –NO2, -
CN etc. which increase the ionization potential, decrease the relative abundance of the
aromatic compounds.

(f) Absence of molecular ion peak in the mass spectrum means that the compound under
examination is highly branched of tertiary alcohols.
(g) In case of chloro or bromo compounds, isotope peaks are also formed along with
the molecular ion peak. In case of bromo compounds, M+ and M++ 2 peaks are
formed in the intensity ratio 1:1. In case of chloro compounds, M+ and M+ + 2
peaks are formed in the intensity ration 1: 3.

 Bromine:
◼ M+ ~ M++2 (50.5% 79Br/49.5% 81Br)

2-bromopropane
M+ ~ M+2
Instrumentation Mass
Analyzer
Heating coil
A B Electron gun C D Magnetic field

Electron collector
Ion
Sample acceleration
Ionization Chamber
vaporization
Chamber
Chamber

Recorder
Detector

Amplifier
 Basic Concepts
• A mass spectrometer is an instrument that Samples may be introduced in gas, liquid or

produces ions and separates them in the gas solid states.

phase according to their mass-to-charge ratio Type of mass analyzer

(m/e or m/z). Today a wide variety of mass • Magnetic field deflection

spectrometers is available, but all of these share • Double focusing mass analyzer
the capability to assign mass-to-charge values • Quadrupole mass analyzer

to ions, although the principles of operation and • Time of flight

the types of experiments that can be done on • FT-ICR (Fourier transform ion cyclotron

these instruments differ greatly. resonance mass analyzer)

Basically, a mass spectrometric analysis can be envisioned to be made up of the following steps:
Sample Introduction → Ionization → Mass Analysis → Ion Detection/Data Analysis
Instrumentation Mass
Analyzer
Heating coil
A B Electron gun C D Magnetic field

e- +
M M
m1+ +
m2
Electron collector
Ion
Sample acceleration
Ionization Chamber
vaporization
Chamber
Chamber

Recorder
Detector

Amplifier
Mass Spectrometry
1. At a given potential (1 – 10 kV) each ion will have a kinetic energy:
m = mass of ion
½ mv2 = eV……(1) v = velocity
V = potential difference
e = charge on ion

As the ions enter a magnetic field, their path is curved; the radius of the
curvature is given by:
r = mv ……(2) H = strength of magnetic field
eH r = radius of ion path
If the two equations are combined to factor out velocity:

m/e = H2r2
2V
Ionization technique
Mass spectrum: Mass spectrum of a compound is significantly depending upon the ionization methods.

• Variation in the spectrum is introduced in terms of the intensities of the signals at various m/e values.
It is highly characteristic of a compound. No two compounds can have exactly similar mass spectra.

• Ionization technique can be categorized into two parts

➢ Hard ionization technique

✓ High energy, increased fragmentation

➢ Soft ionization technique

✓ Low energy, decreased fragmentation

 Ionization methods

Gas Phase Desorption Evaporation

Electron Ionization Field desorption Thermospray APCI

(MALDI) Matrix
Assisted Laser
Chemical Ionization Desorption ESI (Electro
Spray APPI
Ionization
Ionization )
FAB (Fast atom bombardment)
 Electron Bomb Ionization ( EI )
e-
• Sample is heated and energized by a beam of

M
BOOM
electrons, usually gives a molecular ion (M+) and
a lot of fragments due to high energy. So, it is
Hard ionization.

• It is also known as electron bombardment

ionization.

• Electron ionization leads to fragmentation of the

molecular ion, which sometimes prevents its
detection.

• This is the oldest and most popular methods

 Chemical ionization (CI)
• Chemical ionization is a gaseous phase ionization method, it is very important soft ionization technique

• Fragment is less and gives intense peak of molecular ions. Consequently, chemical ionization is complementary
to electron ionization.

• Some molecules like alcohol, ethers, amines, esters are highly fragmented in electron ionization, so molecular
ion peaks will not be detected. For this, we can use CI.

Disadvantages:
Step of CI:
• Need Volatile Sample
A carrier gas/Reagents gas is introduced into the ionization source • Need Thermal Stability
at slightly high pressure. • Quantification Difficult
Carrier gas: methane, ammonia or isobutane • Low Mass Compounds (<1000 amu)
• Solids Probe Requires
• Skilled Operator
Chemical ionization (CI)
2. Primary ions will be reacted with excess CH4 and it
1. Carrier gas will be ionized due to the electron
will produce different type of secondary ions
impact from the ionization source
CH4 +ꙩ + CH4 CH5 + + CH3 ꙩ

Secondary ions
CH4 + e- CH4 +ꙩ + 2e- CH3 + + CH4 C2H5 + + H2
Radical molecular ion
CH4 +ꙩ C2H5 + + CH4 C3H5 + + 2H2
CH3 + + Hꙩ

Primary ions

3. Secondary ions will be reacted with analyte +

• Hydride transfer M + C2H5 + M-1 + C2H6
molecule and form ions by 3 ways
+
+
• Proton transfer M + CH5 + M-H + CH4 +
+ • Electrophilic addition M + CH3 M-CH3
M + C2H5 + M-H + C2H4
 Potential difference
Field Desorption between this anode
and the cathode is
• It is a type of desorption ionization technique which 107 to 108 V/cm

Mass Analyzer
involves direct conversion of solid/liquid molecules into Cathode

gaseous ion directly. M+ +e- M+

+ +
• In this technique low volatile samples are used to M M-H M-H
Intense
produce stable molecular ions electric
field Carbon microneedles
• Samples are loaded on the surface of the carbon
microneedles by dipping in the sample solution. Anode Tungsten
• Carbon microneedles will produce high gradient voltage metal
emitter
on their tips, that’s why sharp tips are used.

Ion formation takes place by two mechanism M M+ +e-

Field ionization electrons are removed from the species/analyte in high electric field.
H+ or Na+
Cation attachment cation will be attached with the analyte molecule, +
M + H+ M-H
Fast Atom Bombardment (FAB)
• FAB is a soft ionization method classified under desorption method.

• FAB is used to determine the molecular weight of the compounds having the size
from 300 to 6000 Daltons.

• Generally used to determine the molecular weight of peptides

Methodology
Characteristics of the matrix
✓ It should be non-volatile

+ ✓ It should be low vapor pressure liquids

Sample ✓ Examples, Glycerols, thioglycerols, 3-nitrobenzyl alcohol
Matrix Sample Matrix
Mixture
 Neutral atom production
FAB Electron gun accelerated

e-

Mass Analyzer
Xe ꙩ+ Xe
Xe Ar ꙩ+ Ar (M+H)+

Ar
Xe ꙩ+ Xe
Ar ꙩ+ Ar
Ionization Acceleration Sample Matrix
chamber by 6 to 10 Probe
Mixture by transitional
keV energy
Xe+e- Xe ꙩ+ +2e- Xe ꙩ+
Ar+e- Ar ꙩ+ +2e- Ar ꙩ+

ꙩ+
Xe, Ar (accelerated neutral atom ) will be bombarded
Xe + Xe ꙩ+ Xe + Xe Removed by the
ꙩ+ to the sample matrix mixture and ionize the sample
Ar + Ar ꙩ+ Ar + Ar electric field
 Matrix Assisted Laser Desorption Ionization (MALDI)
• It is a soft ionization technique under desorption ionization
…… Co-
methods which uses pulsed LASER (Light Amplification …… ………… crystallization
…… + …… ..………
by Stimulated Emission of Radiation) beam. …. …. ….
Solution A Solution B
• It is used to determine the molecular weight of peptides, Solution Crystallization
A+B
antibodies, protein molecules etc. upto the size of 300
Kda. MALDI is achieved in two steps.

• Sample is dissolved in solvent Solution A • In the first step, the compound to be analyzed is

• Matrix is dissolved in solvent dissolved in a solvent containing in solution small

Solution B
organic molecules, called the matrix.
Matrix Materials: nicotinic acid, dihydroxy benzoic acid,
• The second step occurs under vacuum conditions
cinnamic acid derivatives.
inside the source of the mass spectrometer.
Matrix materials should have low molecular weight, acid in
Sample is co-crystallized with a matrix and then
nature, polar functional groups, strong absorption capacity irradiated with laser.
(MALDI)
Different kind of LASER Beams
• 337nm: Nitrogen LASER of UV range LASER beam
+
• 355 nm: Frequency tripled Nd: YAG (Neodynium: Yttrium MH
aluminium garnet)
• 326 nm: Frequency Quadrupled Nd: YAG
• 2.94 μ m: IR LASER produced by Er:YAG

• LASER Beam will hit the samples matrix mixture and

analyte, or sample will be converted into the form of gas.

+
• Protonation M + H+ MH
• Deprotonation M M-H
-
+ H+
 Thermospray Ionization
• It is evaporation ionization method in which solvent molecules
Spray
are removed from the analyte by evaporation. containing
analyte +
• This technique is very popular for coupling with chromatography Heating coil solvent
like HPLC, RP-HPLC etc.
• Eluent sample coming -;from the column will be passed through

………….
the heated capillary tube.
M
• Heated capillary tube will nebulize the eluent, partially evaporate ………………………....……….. M+

the solvent and form a stream of fine spray containing analyte

Heating
• Fine spray droplets will be partially charged by thermal energy capillary
tube
and solvent gets evaporated from analyte. Heating coil

• Due to the thermal energy analytes will be partially ionized and

• Molecular ion M+
solvent will be evaporated +
• Protonation M + H+ MH
• Ionization analytes will move towards the mass analyzer for
• Deprotonation M M-H
-
+ H+
further analysis
Electron spray Ionization (ESI)
 Electron spray Ionization (ESI) ESI
capillary

• It is evaporation ionization technique used to analyze the

Nebulization
high molecular weight biomolecule, labile and non- gas N2 SH+
N2 M
volatile compounds.

Mass Analyzer
Sample Evaporation
• Due to ESI technique mass spectroscopy becomes very +
inlet ………………………....……….. + +
+ ++
popular and generally couples with chromatography. Solvent MH+
• A solution containing the sample molecule is sprayed +analyte

through the high voltage potential capillary by the help N2 +ve -ve

of nebulization gas Heated desolvation gas

Ionization
• Sprayed droplets are ionized due to high voltage Power supply

potential at capillary • It can also produce multiple charged ions along with
single charged ions
• Heated disolvation gas will evaporate the solvent and it
• This techniques is used to ionize peptides, proteins, lipid
will produce the molecular ion or Quasi Molecules Ion.
oligosaccharides, synthetic polymers etc
ElectroSpray Ionization (ESI)
Types of Ions and Peaks in MS
• Molecules Ion / Parent Ion:
Ion formed by the loss of single election at lowest ionization potential from a molecule

• Fragment Ion/ Daughter Ion +

Generated by the fragmentation of Molecules ion in the ionization charter M + H+ MH (M+1)
+
• Quasi Molecules Ion M M-H + H (M-1)
A protonated molecules ion OR An ion formed by removal of one hydrogen atom from molecular ion is
known as quasi molecular ion.

Base peak
-The most intense/ tallest in the mass spectrum M+
-It is due to the greatest relative abundance Base peak M+
Types of Mass Analyzers

➢ Magnetic field deflection

➢ Double focusing mass analyzer

➢ Quadrupole mass analyzer

➢ Time of flight

➢ FT-ICR (Fourier transform ion cyclotron resonance mass analyzer

Magnetic field deflection
Double Focusing Mass Analyzer Magnet
Slit B Electrostatic analyzer
• It gives higher resolution compassed to magnetic
deflection mass analyzer, because electrostatic analyzer is
added to improve the performance.
• Positive charged ions will enter the magnetic analyzer
Slit C
through slit A and defected just like magnetic mass
Ions
analyzer. M+ m1+ m2+
(100) (60) (40)
Slit A Negative Detector
• Magnetic analyzer will separate the ions according to the Magnetic analyzer electrode
deflection in the magnetic field as per the mass differences • Velocity of ions having same m/z or m/e value will
between the ions. m1+ M+ be same under electrostatic analyzer. Peaks are
m2 + m2 + m1+ M+
more then Io times higher resolved as compared to
normal magnetic deflection analyzer.
• When positive ions will be entered into the electrostatic • Now a days double focusing mass analyzer is
analyzer then all ions will be analyzed. used only in the double focusing mode.
Quadrupole mass analyzer

• It consist of four cylindrical metal rods analyzer in a

square parallel to the direction of ion beams.
• Hyperbolic can also be used.
• Radiofrequency (RF) or Direct Current (DC) voltage m1+ M+
is applied. m2+
• Combination of RF or DC will generate oscillating
electrotactic field between the region of rods. • Ions will enter the mass analyzer

Two +ve rods will be • Depending on the ration of RF amplitude and DC voltage
+ve diagonal to each other oscillating electrotactic field will be generated for ions.
-ve -ve
• If RF˃ DC, then the larger ion will hit the detector first M+ m1+ m2+
+ve Two -ve rods will be
diagonal to each other • If DC˃ RF, then the smaller ion will hit the detector first
m2+ m1+ M+
Quadrupole mass analyzer

• Ions will acquire oscillating by two ways

✓ If inappropriate m/z ratio of ions (other, then 1 to 1000) is

present then they will undergo on unstable oscillating and
hit the rod; and ultimately will not reach to the detector.
✓ Ions with correct m/z ration will undergo a stable
oscillating in corkscrew path and strike the detector and
gives signal.
 Time of Flight Mass Analyzer (TOF)
LASER Acceleration
• TOF is based on the simple idea that the Ionization chamber Drift path
chamber
velocities of two ions varies depending on the

Detector
mass of the ions.
• Ions should have created at same instant and
Matrix
+- -
M+
should have the same kinetic energy. m1+
m2 +
• Lighter ions have higher velocity as compared + --
to the heavier ion.
• If ions are travelling towards the detector then
Sample Flight tube
lighter ion will strike the detector first due to Sample
holder Positive Grid
Negative
higher velocity.
Grids
TOF
LASER Acceleration
• Kinetic energy of an ion accelerated through an electrical Ionization chamber Drift path
potential will be chamber
ZV = 1/2mv2 ........................(1)

Detector
+- -
• Velocity of the is the pathlength divided by time (t) Matrix
v=L/t …………. (2) M+
• Put the value of v from eq.(2) to eq.(1) m1+
ZV=1/2mL2/t2 ……………… (3) m2 +
• Rearrangement of eq.(3) + --
m/Z = 2Vt2/L2………… (4) K=2V/L2… (5)
m/Z = Kt2……..(6)

m/Z ∞ Kt2……..(7) Sample Flight tube

Sample
holder Positive Grid
Negative
 Acceleration
TOF Ionization
LASER
chamber Drift path
• m/z of the ions that strike the detector after chamber

travelling the path length (L) is directly

Detector
proportional to the square of the time. Matrix
+- -
M+
• Distance b/w l as accelerating of L (Path
m1 +
m2+
Length). + --

• Ions required to TOF must be created in

short and well defined pulses, so MALDI Sample Flight tube
Sample
used because it suits the condition. Positive Grid
holder Negative
Grids
Mass Techniques

Chromatography: Separation

Mass: Detection

Chromatography-Mass Spectroscopy :
Separation + Detection 43

57
29 71
15 85
99 113 142
GC-MS LC-MS m/z
GC-MS
Gas chromatography-mass spectrometry (GC-MS) is a method that combines
the features of gas-liquid chromatography and mass spectrometry to identify
different substances within a test sample.

HEWLETT Mass
5972A Selective
PACKARD Detector

1.0
DEG/MI
N

MS
HEWLETT
PACKARD

5890

Sample Gas Chromatograph (GC) Mass Spectrometer

B
D A
A C B
D
A C
B
C
DB
A
C D

Sample Separation Identification

GC-MS

GAS CHROMATOGRAPHY MASS SPECTROMETRY

LC-MS
Liquid chromatography-mass spectrometry (LC-MS) is an analytical
chemistry technique that combines the physical separation capabilities of
liquid chromatography with the mass analysis capabilities of mass
spectrometry.

Different compounds exit Identification of each molecule

at different time ion

LC MS
B
Peak A: mass1
A Peak B: mass2
C
Peak C: mass3

t/min
LC-MS

Liquid chromatography-mass spectrometry (Ion trap LCMS system )

 Exercise 1:

Analysis shows exact mass of compound A is 136.0886 and the formula of this
compound is C9H12O, please confirm the structure of compound A.

Answer

100 107
m/z: 136 M-18 M-H2O
% OF BASE PEAK

79 m/z: 118
77
51 77
50
107 M-29 M-C2H5
41 136
39 118
-C2H5

0 20 40 60 80 100 120 140 160 H2

C CH
CH 3

OH

51
Continue with
Part-ll