MAEERS MAHARASHTRA INSTITUTE OF

PHARMACY, PUNE
A
SEMINAR ON MASS SPECTROMETRY
As per Pune University syllabus 1
st
semester

Presented By: Mr. Patil Somnath Ashok

Guided By: Principal Dr. B.S. Kuchekar
1
CONTENTS:
Introduction
Definition
Classification
Atomic MS
Molecular MS
Principle of MS
Instrumentation
Ion sources
Mass analyzers
Detectors
Vacuum system
Applications


2
Introduction:
In Mass Spectroscopy (MS), atomic and
molecular weights are generally expressed in
terms of atomic mass units (amu). The atomic
mass unit is based upon a relative scale in
which the reference is the carbon isotope
12
C
6
, which is assigned a mass of exactly 12
amu. Thus the amu is defined as 1/12 of the
mass of one neutral carbon atom.
3
Mass Spectroscopy
Mass spectroscopy is perhaps one of the
most widely applicable of all the analytical
tools available to the analytical chemist in
the sense that this technique is capable of
providing information about :
4
the qualitative and quantitative composition
of both organic and inorganic analytes in
complex mixtures
this instrument measures compounds with
molecular masses up to 200, 000 Daltons.
the structures of a wide variety of complex
molecular species
isotopic ratios of atoms in samples and
the structure and composition of solid
surfaces.
5
Mass Spectroscopy
Molecular Mass

Spectroscopy
Atomic Mass

Spectroscopy
6
Atomic Mass Spectrometry
Nearly all elements in the periodic table
can be determined by mass
spectrometry
More selective and sensitive than
optical instruments
Simple spectra
Isotope ratios
Much more expensive instrumentation
7
Atomic Mass
Spectrometry (Inorganic MS)

Mass Spectrometers
ICP-MS
Spark Source MS
Glow-Discharge MS
Elemental Surface Analysis by MS
Laser Ablation ICP-MS
8
Atomic Mass Spectrometry processes:

Atomization (sample intro)
Conversion to ions
Separation based on m/z ratio
Detection

In other forms of MS (GC-MS or MS of organic compounds), sample
introduction does not involve making atoms, just getting molecules
into the high vacuum system
9
Advantages of Atomic Mass Spectrometry
over Optical Atomic Spectrometry:
1) Detection limits are better, sometimes several orders of
magnitude
2) Very simple spectra
3) Ability to measure isotope ratios
Disadvantages:
1) Equipment cost
2) Instrument drift
3) Isotopic interferences
10
Advances In Atomic Mass Spectrometry

11

12
Principle
Mol. Are bombarded with a beam of energetic electron

Ionization of mol. & fragmentation

Some of which are +ve

Each ion has a particular ratio of mass to charge i.e. m/e
ratio

For most of ion charge is 1

So m/e =mol. Wt of ion.
13
Mass Spectrometry
It subjects vaporized molecules to bombardment by
a stream of high-energy electrons, converting these
molecules to ions
These ions are then accelerated in an electric field
The accelerated ions are then separated according to
their mass-to-charge ratio in a magnetic or electric
field
The ions that have a particular mass-to-charge ratio
are then detected by a device that counts the
number of ions striking it.
Simplest form of mass spectrometer performs 4 essential
functions: (under vacuum 10
-6
mm Hg)
14
Positive or Negative Ion Mode?
If the sample has functional groups that readily
accept H
+
(such as amide and amino groups
found in peptides and proteins) then positive ion
detection is used-PROTEINS

If a sample has functional groups that readily
lose a proton (such as carboxylic acids and
hydroxyls as found in nucleic acids and sugars)
then negative ion detection is used-DNA
15
INSTRUMENTATION:
System
Inlet
Ion
Source
Mass
Analyzer
Detector

Vacuum
System

Signal
processor
m/z
10
-5
to 10
-8
Torr
MASS SPECTROMETER
16
Instrumentation available:

VG Analytical 70-250-SE Normal geometry double focusing MS.
Micromass TofSpec2E Reflectron MALDI-TOFMS.
ThermoQuest TraceMS Single quadrupole GC-MS.
Bruker ApexIII FT-ICR-MS.
ThermoBioAnalysis Dynamo Linear MALDI-TOFMS.
Micromass Platform II Open access single quadrupole MS.
Waters ZMD Open access single quadrupole MS.
ThermoQuest TraceMS Open access single quadrupole GC-MS.
ThermoFinnigan LCQ Ion trap.
Micromass Platform II Single quadrupole MS

17
Resolution & Resolving Power

Width of peak indicates the resolution of the MS
instrument

The better the resolution or resolving power, the
better the instrument and the better the mass
accuracy
Resolving power is defined as:

M is the mass number of the observed mass (DM) is
the difference between two masses that can be
separated

18
M
M

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49
mass
0
100
%
36.5
e.g. the mass spectrum of HCl
19
25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49
mass
0
100
%
Scan EI+
7.58e12 36.0
38.0
20

1. Sample Introduction:

All Glass Heated Inlet System (AGHIS):
Direct Insertion Probe (DIP):
MALDI pattern
Direct Inlets:
Gas chromatography column
API interface
GC, HPLC,
21
The Sample Inlet System
Batch Inlet Systems:

simplest and involve
volatilization of liquid
For gases, introduced
into the metering
volume container and
expanded into the
reservoir flask

The Direct Probe Inlet:

used for solids and non-
volatile liquids

sample is introduced by
means of a sample holder, or
probe,

the amount of the sample
to be analyzed is small.
22
2. IONISATION SOURCES
CLASSIFICATION:



GAS PHASE ION SOURCE: HARD SOURCE:
DESORPTION ION SOURCE: SOFT SOURCE:




23
1. Gas Phase:
1. Electron ionization (EI)
2. Chemical ionization (CI)
3. Field Ionization (FI)
2. Desorption
1. Field Desorption (FD)
2. Fast Atom Bombardment (FAB)
3. Secondary Ion Mass Spectrometry (SIMS)
4. Laser Desorption (LD)
5. Plasma Desorption (PD)
6. Thermal Desorption
7. Thermo spray ionization (TS)
8. Electrospray (ES)

24
Gas-Phase Sources
Gas-phase sources require volatilization of the
sample before ionization and thus are limited
to thermally stable compounds that have
boiling points less than about 500C.
25
ELECTRON-IMPACT SOURCE
Ion sources for gases:
26

Electron Impact Source : In the sources,
electrons emitted from a filament are accelerated
by a potential of about 70 V and made to collide
with gaseous atoms or molecules of the sample
causing ionization. Electron-impact ionization is not
very efficient and only about one molecule in a
million undergoes the primary reaction
M + e- = M.
+
+ 2e-
27
Electron Impact
Advantages
Well-Established
Fragmentation Libraries
Insoluble Samples
Interface to GC
Non-Polar Samples


Disadvantages
Parent Identification
Need Volatile Sample
Need Thermal Stability
No Interface to LC
Low Mass Compounds
(<1000 amu)
Solids Probe Requires
Skilled Operator
28
Chemical ionization:
Transfers less energy, produces less fragmentation
Introduce small molecule e.g. CH
4
, NH
3
, C
4
H
10
into closed
ion source, 1 Torr, as reagent, R, to produce electron-
impact ionization.
R is selective.
e.g. E.I.: CH
4
+ e CH
3
+
, CH
4
+
+ 2e
then 2
ary
ions: CH
4
+
+ CH
4
CH
5
+
+ CH
3
or CH
3
+
+ CH
4
C
2
H
5
+
+ H
2

(i.e. large amount of gas R, small amount of sample) 29
Behavior of R :
1. As strong acid (proton transfer):
e.g. AH + CH
5
+
AH
2
+
+ CH
4

analyte protonated

2. Hydride ion transfer (H
-
abstraction):
C
2
H
5
+
+ AH A
+
+ C
2
H
6

fragmentation less extensive than with E. I.
3. Charge transfer:
CH
4
+
+ AH CH
4
+ AH
+


30
Main differences C. I. & E.I.
1. C.I. reagent gas produces an ion that
protonates analyte
M MH
+
; AH AH
2
+
These ions, with paired e, more stable
than radical species from E.I
2. Pattern of fragmentation in C.I. is
complementary to that in E.I.
C.I. No molecular ion, but MH
+.

Simple spectra. Easy to identify mixtures.
31
3. Degree of fragmentation in C.I. (F.I.) smaller.
M.S. more easily interpreted. E.I. wealth of fragments at
expense of M
+
.

4. C.I. more sensitive than E.I.
Ionization concentrated in few ions.
For E.I. ionization efficiency is 0.1%.
Residence time inc. & pressure of R inc. give more ions in
C.I.

32
Chemical Ionization
Advantages

Parent Ion
Interface to GC
Insoluble Samples

Disadvantages

No Fragment Library
Need Volatile Sample
Need Thermal Stability
Quantitation Difficult
Low Mass Compounds
(<1000 amu)
Solids Probe Requires
Skilled Operator
33
Field ionization source:
34

1. Activate wire anode with sharp points, whiskers, 10-10
2
nm radius,
by filling chamber with C
6
H
5
CN at 0.04-0.1 Torr, apply 10
4
V for 2h.
(or just use sharp-pointed metal anode).

2. Introduce sample
Some deposits on anode tip.

3. Anode is gun input, 10
5
V/cm
between anode & cathode, under high vac.

4. Electric field sufficient to remove e
-
, but not excite M
+


Little fragment
n
(no e
-
collisions; ions move into analyzer in 10
-6
,
10
-7
s). M
+
and (M+1)
+
abundant.
About 10 less sensitive than E.I. 35


36
The matrix chromospheres absorbs and
distribute the energy of a laser, produced a
plasma,
vaporizes and ionize the sample.
+ rapid, convenient for molecular weight
(singly charged ions mostly)
- MS/MS difficult, almost not compatible
with LC coupling
<500000Dac
Matrix materials used are: benzoic acid der. ,
cinnamic acid der. & nicotinic acid der.s
37
MALDI
Advantages

Parent Ion
High Mass Compounds
(>100,000 amu)
Thermally Labile
Compounds (R.T.)
Easy to Operate
Disadvantages

No Fragment Library
Wide variety of matrices
Quantitation Difficult
38
2.Fast Atom Bombardment (FAB)
The sample is dissolved in a liquid matrix such as glycerol, thioglycerol,
m-nitro benzyl alcohol, or diethanolamine and a small amount (about
1 micro liter) is placed on a target.
The target is bombarded with a fast atom beam (for example, 6 keV
xenon atoms) that desorbs molecular-like ions and fragments from the
analyte.
Cluster ions from the liquid matrix are also desorbed and produce a
chemical background that varies with the matrix used.
Xe
e
-

Xe
+
+ Xe

Xe
+
Is accelerated to ~6-10 keV, and pass through Xe
Xe + Xe
+

Xe (Xenon with Kinetic energy) FAB
39
Benefits:
Sample introduction can be through direct
insertion probe or LC/MS (continuous-flow FAB).
rapid, simple
relatively tolerant of variations in sampling
good for a large variety of compounds
Useful fragmentation pattern
strong ion currents -- good for high-resolution
measurements
40
Limitations:

high chemical background defines detection limits
may be difficult to distinguish low-molecular-weight compounds
from chemical background
analyte must be soluble in the liquid matrix
no good for multiply charged compounds with more than 2 charges
requires a high concentration of the organic liquid matrix (typically
80 to 95% glycerol) which limits sensitivity
Mass range
Moderate Typically ~300 Da to about 6000 Da.
41
3. Thermal desorption
Coat sample on thin wire insertion probe. Heat 100-200
o
C s
-1
to get
desorption/evap
n
rather than decomp
n
. Then use EI, CI etc. Use for inorganic
compounds with low I.E. 3-6 eV.

4. Field desorption
Coat field ioniz
n
. emitter (carbon needle) with nonvolatile sample. Heat wire & apply
high electric p.d. Useful for studying surface phenomena such as adsorbed or
trapped species.
5.
252-
Plasma desorption
10-50 cm
252
Cf to minimize fragmentation
D ~m/2 ~m/2 electronic excitation accelerates
set localized plasma of molecule
t = 0 ~75MeV & fragment ions
charge < 20 sample charged
film on grid D
metal foil

Low ion yield, long counting time, calibration m/z from t for Na
+
but up to 20000amu.
Deposit sample on foil and bombard with fission fragments of MeV energy.
42
6. ESI: sample solution sprayed across high
p.d. from a needle into an interfaced
orifice. Heat and flow gas desolvate the
charged droplets, producing multiply-
charged ions. Used for charged, polar or
basic compds. up to 50 kDa.

LC
N
2
(g)
+
-
to rotary pump:
1 mbar
to high vacuum pump:
10
-4
-10
-5
mbar
to ms
nebulizing gas
43
ESI
Advantages

Parent Ion
High Mass Compounds
(>100,000 amu)
Thermally Labile
Compounds (<0 C)
Easy to Operate
Interface to HPLC
Zeptomole sensitivity with
nanospray
Disadvantages

No Fragmentation
Need Polar Sample
Need Solubility in Polar
Solvent (MeOH, ACN, H
2
O,
Acetone are best)
Sensitive to Salts
Suppression
44
Types of ions produced in MS
Molecular ion/Parent ion
Daughter ion
Rearrangement ion
Multiple charged ion
Negative ion
Metastable ion
45
Components of MS:
1.Mass Analyzers
Mass
Analyzer
Dispersion of the ions is based on mass-to-charge ratio
There are several type of mass analyzers.
1. Magnetic sector
2. Electrostatic and Magnetic sector
3. Quadrupole MS filter
4. Ion trap analyzers
5. TOF Time-of-Flight
6. FT-MS
46
2. Detector:
Detector
Convert the beam of ions in an electrical signal that can be
processed, stored, displayed and recorded in many ways.

Vacuum
System

3.Vacuum:
MS require the high vacuum is maintained in all
spectrometer components (except signal processing)

Electron Multiplier (most commonly used)
Faraday cup
Photographic plates
Scintillation type
Other:

47
Vacuum Systems
Reduce gas molecules in analysis chamber
Ion detectors need low pressure environment
Dont want to detect gas in system
Want to detect analyte before it can collide or
react with gas in system
Surface analysis: a need for analyzing an
unoxidized surface
48
Vacuum Pump Selection
Atmosphere to 10
-2
Torr
Roughing pumps
10
-4
10
-8
Torr
Diffusion pumps
Turbo molecular pumps
Cryogenic pumps
10
-9
10
-12
Torr
Ion pumps

49
Vacuum Regions
Rough Vacuum
760-1 Torr
Mid Vacuum
1 10
-3
Torr
High Vacuum
10
-3
10
-7
Torr
Ultrahigh Vacuum (UHV)
<10
-7
Torr
50


Magnetic Sector Analyzers:
51
Magnetic sector analyzers employ a permanent magnet or
electromagnet to cause the beam from the ion source to
travel in a circular path of 180, 90, or 60 degrees. Here, ions
are formed by electron impact.
The translational energy of an ion of mass m and charge z
upon exciting slit B is given by
K = ZeV = mv
2


where V is the voltage between A and B, v is the velocity of
the ion after acceleration, and e is the charge of the ion.
The path in the sector described by the ions of a given mass
and charge represents a balance between two forces acting
upon them. These two forces are the centripetal force and
the magnetic force and equating these two forces yields:
Bzev = mv
2
/r which rearranges to v = Bzer/m
Substituting the above equation into (1) gives m/z =
B2r2e/2V

52
Double Focusing Instruments:
53
These type of instruments, unlike single-
focusing which simply minimize
directional errors, are designed to limit
both the errors introduced because ions
are initially moving in different
directions and also the errors
introduced due to the fact that ions of
the same mass-to-charge ratio may have
different translational energies.
54
The characteristics are listed below:
Classical shaped mass spectra
Very high reproducibility
Best quantitative performance of all mass spectrometer
analyzers
High resolution
High sensitivity
High dynamic range
Linked scan MS/MS does not need another analyzer
High energy CID (collision induced decay) MS/MS spectra are
very reproducible
55
The limitations of sector instruments can be
summarized as:

Not well-suited for pulsed ionization methods (e.g.
MALDI)
Usually larger and higher costs both in purchase and
maintenance than other mass analyzers
Linked scanning MS/MS gives either limited precursor
selectivity with unit product-ion resolution or nominal
precursor selection with poor product-ion resolution
56
Applications:

All organic MS analysis methods
Accurate mass measurements/peak-matching
Quantitation
Isotope ratio measurements
57
Time-of-Flight analyzer (TOF)
58
In TOF instruments, positive ions are produced periodically using
brief pulses of electrons,
secondary ions or
laser generated photons
These pulses have typically a frequency of 10-50 kHz and lifetime
0.25ms
The ions produced are then accelerated by electric field pulse (10
3
-10
4

V) that has the same frequency as ionization pulse but lags behind.
The accelerated ions then pass into a field-free drift tube (about 1 m
long)
59
Advantages

Extremely High Mass Range
(>1 MDa)
Fast Scanning
Disadvantages

Low Resolution (4000)
Low Accuracy (>200ppm)
MS/MS not possible
Time-of-Flight (TOF)
60


Quadrupole Mass Analysers:
61


62







63

The ion trap functions both as an analyser and an ion
storage device. The ions are produced or introduced into
the ion trap, trapped and can be progressively ejected form
the ion trap volume by ramping of the rf voltage. The
trapped ion can also be induced to undergo dissociation
and hence produce the MSMS spectrum within the one
analyser. Note the difference with FT-ICR-MS where the
frequency of the circulating ions is measured and Fourier
Transformed in give the MS.
64
Reflectron Time-of-Flight (TOF)
65
Advantages

High Resolution (>20,000 in
some models)
High Accuracy (<5ppm)
10,000 Mass Range
Fast Scanning
Disadvantages

Low Resolution for MS/MS
(PSD)
Reflectron Time-of-Flight (TOF)
66
FT-ICR-MS
67
FT-ICR
Fourier-transform ion cyclotron resonance
Uses powerful magnet (5-10 Tesla) to create a
miniature cyclotron

Originally developed in Canada (UBC) by A.G.
Marshal in 1974

FT approach allows many ion masses to be
determined simultaneously (efficient)

Has higher mass resolution than any other MS
analyzer available

68
Detectors:
Two common types of detectors:
1) Faraday Cup
2) Electron Multiplier

Faraday Cup:
Ions are accelerated toward a grounded collector electrode As
ions strike the surface, electrons flow to neutralize charge,
producing a small current that can be externally amplified. Size
of this current is related to the number of ions .
less sensitive
69
70
Electron Multiplier:
Analogous to PMT
Durable, applicable to
most analyzers
Ions strike surface of
dynode
Generate electrons
Ejected electrons are
accelerated to other
dynodes
Current is related to
number of ions.
71
APPLICATIONS:
1. Elucidation of the structure of organic and
biological molecules.
2. Determination of molecular mass of peptide,
proteins and oligonucleotides.
3. Identification of components in thin layer and
paper chromatograms.
4. Determination of amino acids sequences in sample
of polypeptides and proteins.
5. Detection and identification of species separated
by chromatography.

72
6. Identification of drugs of abuse and metabolites of
drugs of abuse in blood, urine, and saliva.
7. Monitoring gases in patients breath during
surgery.
8. Testing for the presence of drugs in blood in race
horses and in Olympic athletes.
9. Analyses of aerosol particles.
10. Determination of pesticide residues in food.
11. Monitoring volatile organic species in water
supplies.
73
Conclusions
Mass spectrometers exist in many different
configurations to allow different problems to be
solved
All mass spectrometers have a common
architecture and relatively similar operating
principles
Understanding the applications and limitations
of MS in proteomics will help in understanding
and meeting the bioinformatics needs in
proteomics
74
Bibliography:
Instrumental Analysis By Skoog, Holler, Crouch 8
th
Indian
Reprint 2010 Page No. 318 & 606
Instrumental Methods Of Chemical Analysis By G.R. Chatwal &
S.K. Anand 1
st
Edition 1979 Page No. 2.272
Introduction to spectroscopy by Donald L. Pavia Gary M.
Lampman George S. Kriz 3
rd
edition page no. 390
www.studytemple.com
http://www.spectorscopymag.com/spectrbl.htm
http://www.organicchemistryonline.com/OCOL.HTM#
http://www.scimedi.com/chem-ed/analytic/ac-meths.htm
http://masspecscripps.edu/hist.htm
http://www.hdscience.com/
http://www.scimedia.com/chem-ed/ms/ms-intro.htm

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