Mass spectrometry

By
Kommineni.vidyachowdhary M.pharm
Vaagdevi pharmacy college
warangal
1
MASS SPECTROMETRYMASS SPECTROMETRY

Mass spectrometry is the most accurate method for
determining the molecular mass of the compound and its
elemental composition.
It is also called as positive ion spectra or line spectra.
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2

Introduction
• Mass spectrometry (Mass Spec or MS) uses high energy
electrons to break a molecule into fragments.
• It does not involve the absorption or emission of light.
• A beam of high-energy electrons breaks the molecule apart.
• The masses of the fragments and their relative abundance
reveal information about the structure of the molecule.
• Separation and analysis of the fragments provides information
about:
– Molecular weight
– Structure

Mass Spec Principles
IonizerIonizer
Sample
+
_
Mass AnalyzerMass Analyzer DetectorDetector

06/13/15 5
Schematic of Mass Spectrometry
Ionizer
↓
Mass-to-charge ratio Analyzer
↓
Detector

How does a mass spectrometer work?
• Ionization
method
– MALDI
– Electrospray
(Proteins must be charged
and dry)
• Mass analyzer
– MALDI-TOF
• MW
– Triple Quadrapole
• AA seq
– MALDI-QqTOF
• AA seq and MW
– QqTOF
• AA seq and
protein modif.
Create ions Separate ions Detect ions
• Mass
spectrum
• Database
analysis

06/13/15 7
What is a Mass Spectrometer?
A Mass Spectrometer is a machine that
weighs molecules ! (by measuring the
mass to charge ratio of ions)
Source
EI
CI
ESI
APCI
APPI
MALDI
Dispersion
TOF
FT-ICR
Sector
Quad
Trap
Detector
Faraday Cup
Channeltron
MCP

06/13/15 8
Mass Spectrometry Categorization
• Based on ionization:
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry
Electrospray ionization (ESI) mass spectrometry
Induction-coupled plasma mass spectrometry (ICP-MS)
Atmospheric Ionization Mass Spectrometry
Secondary Ionization Mass Spectrometry (SIMS)
• Based on M/Z Separation:
Quadrupole Mass spectrometry
Ion trap mass spectrometry
magnetic sector mass spectrometry
time-of-flight mass spectrometry
Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
Ion Mobility Mass Spectrometry

06/13/15 9
Mass Spectrometry Categorization
(continued)
• Based on Applications:
Environmental Mass Spectrometry
Biological Mass Spectrometry
Cell Mass Spectrometry
Portable Mass Spectrometry
• Based on Configuration:
Tandem Mass Spectrometry
• Based on Sample Introduction:
GCMS; LCMS, Electrophoresis Mass Spectrometry

06/13/15 10
MS Operation
• Nearly all mass spectrometers need to operate under
high vacuum condition with the pressure less than 10
-5
Torr with the only exceptions of an ion trap mass
spectrometer (milli-Torr) and an ion mobility mass
spectrometer (Torr).
• Never turn on a mass spectrometer without knowing
the chamber pressure.
• A tour to major mass spectrometry facilities in
Genomic Research Center, Sinica will be arranged.

Mass spectrometersMass spectrometersL in e a r T im e O f F lig h t tu b e
R e fle c t o r T im e O f F lig h t t u b e
d e te c to r
re fle c to r
io n s o u rc e
io n s o u rc e
d e te c to r
tim e o f flig h t
tim e o f flig h t
• Time of flight (TOF) (MALDI)Time of flight (TOF) (MALDI)
– Measures the time required for ions to fly down the length
of a chamber.
– Often combined with MALDI (MALDI-TOF) Detections from
multiple laser bursts are averaged. Multiple laser
• Tandem MS- MS/MSTandem MS- MS/MS
-separation and identification of compounds in complex
mixtures
- induce fragmentation and mass analyze the fragment ions.
- Uses two or more mass analyzers/filters separated by a
collision cell filled with Argon or Xenon
• Different MS-MS configurations
– Quadrupole-quadrupole (low energy)
– Magnetic sector-quadrupole (high)
– Quadrupole-time-of-flight (low energy)
– Time-of-flight-time-of-flight (low energy)

LC/LC-MS/MS-Tandem LC, Tandem MS

Typical Mass Spectrum
• Characterized by sharp, narrow peaks
• X-axis position indicates the m/z ratio of a given ion
(for singly charged ions this corresponds to the mass
of the ion)
• Height of peak indicates the relative abundance of a
given ion (not reliable for quantitation)
• Peak intensity indicates the ion’s ability to desorb or
“fly” (some fly better than others)

m/z ratio:
Molecular weight divided by the
Charge on this protein
All proteins are sorted based on a
mass to charge ratio (m/z)

Typical Mass Spectrum
aspirin
RelativeRelative
AbundanceAbundance
120 m/z-for singlysingly charged ion this is the mass

Resolution & Resolving PowerResolution & 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 (∆M) is the
difference between two masses that can be separated

Resolution in MSResolution in MS

Resolution in MSResolution in MS
QTOF
783.455
784.465
785.475
783.6

Introduction to Mass Spectrometry
Sample
introduction
Ionization
Minimize collisions, interferences
Separate
masses
Count ions
Collect results
Nier-type
mass spec

The sample cone isolates the
torch from the interior.
The torch box of an
Agilent 7500 ICPMS
spray chamber
torchAr feed
RF coil

N
SSource
magnets
Filament
Collector
Sample Inlet
+
+ +
+
+
+
++
+
+
+
+
+
+
+
+
Extraction
lenses
Electron ionization
06/13/15
25

In this type anode and cathode are arranged with a very
fine gap (0.5 to 2mm) which may serve as a slit.
The gaseous sample introduced at the anode points where
the electric field is concentrated.
The ionisation of the sample takes place by extraction of
electrons from the sample by microtips of the anode.
06/13/15
26

N
SSource
magnets
Filament
Collector
Sample Inlet
+
+ +
+
+
+
++
+
+
+
+
+
+
+
+
Extraction
lenses
+
++
+
+
+
+
+
+ +
+
+
+
+
Chemical ionization
06/13/15
27

These are the devices used to separate
the ions produced in the ion source into
their individual m/z ratios and focus them
on the detector.
A number of different mass analysers
exist in mass spectrometry.
06/13/15
28

 Magnetic sector – single focussing
- double focussing
 Quadrupole analysers
 ion trap (Quistor) devices
 tiMe-of-flight (tof) analysers
06/13/15
29

Magnetic sectors – single focussing
•Generally bulky and expensive.
•Earliest type of analyser and still popular.
•High accelerating potential especially in
comparison with other methods (usually 4-
10kV).
•Magnets wedge shaped and must provide
homogeneous fields.
•Ion beam enters and exits at exactly 90˚.
•Focuses ions according to their momentum.
06/13/15
30

Magnetic sectors – double focussing
•Adds a second electric sector to provide
energy focussing of ions independent of mass.
•Double focussing means that both the energy
and momentum focus is designed to coincide
at the collector slit.
•Very high mass resolution can be achieved by
this arrangement.
•Very bulky and expensive but high
performance.
06/13/15
32

Quadrupole Mass Analyzer
06/13/15
34
+
+
+
+

The quadrupole consists of four parallel rods. The opposing rods
have the same polarity whilst adjacent rods have opposite polarity.
Each rod is applied with a DC and an
RF voltage.Ions are scanned by
varying the DC/Rf quadrupole
voltages.
Only ions with the selected mass to
charge ratio will have the correct
oscillatory pathway in the Rf field.
06/13/15
35

• Consists of ring electrode
and two end caps
• Principle very similar to
quadrupole
• Ions stored by RF & DC
fields
• Scanning field can eject
ions of specific m/z
• Advantages
- MS/MS/MS…..
- High sensitivity full scan
MS/MS
06/13/15
36

• In this type of analyser the sorting of the ions is
done in absence of magnetic field.
• It operates on the principle that, if the ions
produced are supplied with equal energy and
allowed to travel predetermined distance then
they will acquire different velocities depending
of their masses.
06/13/15
38

• The detector records the charge induced when an ion
passes by or hits a surface
• Electron Multipliers (EM)*
– Most common detector
– -Can Detect positive and negative ions
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39

• Faraday Cup
– Least expensive detector
– Captured ions transfer charge to cup
– used to calibrate other MS detectors
06/13/15
40

• Photographic detection:
This detector system is most sensitive than any
other detector because the photoplate integrates the
ion signal over a period of time.
The photoplates are processed by the usual
photographic techniques and read with the aid of
densitometer.
06/13/15
41

Different Ionization Methods
• Electron Impact (EI - Hard method)
– small molecules, 1-1000 Daltons, structure
• Fast Atom Bombardment (FAB – Semi-hard)
– peptides, sugars, up to 6000 Daltons
• Electrospray Ionization (ESI - Soft)
– peptides, proteins, up to 200,000 Daltons
• Matrix Assisted Laser Desorption (MALDI-Soft)
– peptides, proteins, DNA, up to 500 kD

Electron Impact IonizationElectron Impact Ionization
• Sample introduced into instrument by heating it
until it evaporates
• Gas phase sample is bombarded with electrons
coming from rhenium or tungsten filament (energy =
70 eV)
• Molecule is “shattered” into fragments (70 eV >> 5
eV bonds)
• Fragments sent to mass analyzer

EI Fragmentation of CHEI Fragmentation of CH33OHOH
CH3OH CH3OH+
CH3OH CH2O=H+
+ H
CH3OH +
CH3 + OH
CHO=H+ + HCH2O=H+
Why wouldn’t Electron Impact be suitable
for analyzing proteins?

Why You Can’t Use EI For AnalyzingWhy You Can’t Use EI For Analyzing
ProteinsProteins
• EI shatters chemical bonds
• Any given protein contains 20 different amino acids
• EI would shatter the protein into not only into
amino acids but also amino acid sub-fragments and
even peptides of 2,3,4… amino acids
• Result is 10,000’s of different signals from a single
protein -- too complex to analyze

Soft Ionization MethodsSoft Ionization Methods
337 nm UV laser
MALDI
cyano-hydroxy
cinnamic acid
Gold tip needle
Fluid (no salt)
ESI
+
_

Soft IonizationSoft Ionization
• Soft ionization techniques keep the molecule of interest
fully intact
• Electro-spray ionization first conceived in 1960’s by
Malcolm Dole but put into practice in 1980’s by John
Fenn (Yale)
• MALDI first introduced in 1985 by Franz Hillenkamp and
Michael Karas (Frankfurt)
• Made it possible to analyze large molecules via
inexpensive mass analyzers such as quadrupole, ion trap
and TOF

Ionization methodsIonization methods
• Electrospray mass spectrometry (ESI-MS)
– Liquid containing analyte is forced through a steel capillary at high voltage to electrostatically
disperse analyte. Charge imparted from rapidly evaporating liquid.
• Matrix-assisted laser desorption ionization (MALDI)
– Analyte (protein) is mixed with large excess of matrix (small organic molecule)
– Irradiated with short pulse of laser light. Wavelength of laser is the same as absorbance max
of matrix.

Electrospray IonizationElectrospray Ionization
• Sample dissolved in polar, volatile buffer (no salts)
and pumped through a stainless steel capillary (70 -
150 µm) at a rate of 10-100 µL/min
• Strong voltage (3-4 kV) applied at tip along with flow
of nebulizing gas causes the sample to “nebulize” or
aerosolize
• Aerosol is directed through regions of higher
vacuum until droplets evaporate to near atomic size
(still carrying charges)

Electrospray (Detail)Electrospray (Detail)

Electrospray IonizationElectrospray Ionization
• Can be modified to “nanospray” system with flow < 1
µL/min
• Very sensitive technique, requires less than a picomole
of material
• Strongly affected by salts & detergents
• Positive ion mode measures (M + H)+
(add formic acid to
solvent)
• Negative ion mode measures (M - H)-
(add ammonia to
solvent)

Positive or Negative Ion Mode?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

Matrix-Assisted Laser DesorptionMatrix-Assisted Laser Desorption
IonizationIonization
337 nm UV laser
MALDI
cyano-hydroxy
cinnamic acid

MALDIMALDI
• Sample is ionized by bombarding sample with laser
light
• Sample is mixed with a UV absorbant matrix
(sinapinic acid for proteins, 4-hydroxycinnaminic
acid for peptides)
• Light wavelength matches that of absorbance
maximum of matrix so that the matrix transfers
some of its energy to the analyte (leads to ion
sputtering)

MALDI IonizationMALDI Ionization
+
+
+
+
-
-
-
+
+
+
+
-
-
--+
+
Analyte
Matrix
Laser
+
+
+
• Absorption of UV radiation by
chromophoric matrix and ionization
of matrix
• Dissociation of matrix, phase
change to super-compressed gas,
charge transfer to analyte molecule
• Expansion of matrix at supersonic
velocity, analyte trapped in
expanding matrix plume
(explosion/”popping”)
+
+
+

MALDIMALDI
• Unlike ESI, MALDI generates spectra that have just a singly
charged ion
• Positive mode generates ions of M + H
• Negative mode generates ions of M - H
• Generally more robust that ESI (tolerates salts and nonvolatile
components)
• Easier to use and maintain, capable of higher throughput
• Requires 10 µL of 1 pmol/µL sample

Principal for MALDI-TOF MASSPrincipal for MALDI-TOF MASS
+
+
++
++
+
+
+
+
p u ls e d
U V o r IR la s e r
( 3 - 4 n s )
d e te c to r
v a c u u m
s t r o n g
e le c tr ic
fie ld
T im e O f F lig h t t u b e
p e p tid e m ix tu r e
e m b e d d e d in
lig h t a b s o r b in g
c h e m ic a ls ( m a tr ix )
c lo u d o f
p r o to n a te d
p e p tid e m o le c u le sa c cV

Principal for MALDI-TOF MASSPrincipal for MALDI-TOF MASS
L in e a r T i m e O f F li g h t t u b e
R e f le c t o r T im e O f F l ig h t t u b e
d e t e c t o r
r e f le c t o r
io n s o u r c e
io n s o u r c e
d e t e c t o r
t im e o f flig h t
t im e o f flig h t

MALDIMALDI == SELDISELDI
337 nm UV laser
MALDI
cyano-hydroxy
cinnaminic acid

MALDI/SELDI SpectraMALDI/SELDI Spectra
Normal
Tumor

Background of fragmentation
• The impact of a stream of high energy
electrons causes the molecule to lose an
electron forming a radical cation.
– A species with a positive charge and one unpaired
electron
+ e
-
C H
H
H
H H
H
H
HC + 2 e
-
Molecular ion (M+
)
m/z = 16

Background
• The impact of the stream of high energy electrons can also
break the molecule or the radical cation into fragments.
(not detected by MS)
m/z = 29
molecular ion (M
+
) m/z = 30
+ C
H
H
H
+ H
HH C
H
H
C
H
H
H C
H
H
C
H
H
H C
H
H
+ e
-
H C
H
H
C
H
H
H
m/z = 15

Background
• Molecular ion (parent ion):
– The radical cation corresponding to the mass of the
original molecule
• The molecular ion is usually the highest mass in
the spectrum
– Some exceptions w/specific isotopes
– Some molecular ion peaks are absent.
H
H
H
HC H C
H
H
C
H
H
H

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

Background
• The cations that are formed are separated by
magnetic deflection.

Background
• Only cations are detected.
– Radicals are “invisible” in MS.
• The amount of deflection observed depends on
the mass to charge ratio (m/z).
– Most cations formed have a charge of +1 so the
amount of deflection observed is usually
dependent on the mass of the ion.

Background
• The resulting mass spectrum is a graph of the
mass of each cation vs. its relative 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.

Background
M+
base peak
The mass spectrum of ethanol

Background
• Most elements occur naturally as a mixture of
isotopes.
– The presence of significant amounts of heavier
isotopes leads to small peaks that have masses
that are higher than the parent ion peak.
• M+1 = a peak that is one mass unit higher
than M+
• M+2 = a peak that is two mass units higher
than M+

Easily Recognized Elements in MS
• Nitrogen:
– Odd number of N = odd MW
CH3CN
M
+
= 41
SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced
Industrial Science and Technology, 11/2/09)

 Bromine:
 M+
~ M+2 (50.5% 79
Br/49.5% 81
Br)
2-bromopropane
M+
~ M+2
SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced Industrial Science and
Technology, 11/1/09)

• Chlorine:
– M+2 is ~ 1/3 as large as M+
Cl
M+2
M+

• Sulfur:
– M+2 larger than usual (4% of M+
)
M+
Unusually
large M+2
S

• Iodine
– I+
at 127
– Large gap
Large gap
I+
M+
ICH2CN

Fragmentation Patterns
• The impact of the stream of high energy
electrons often breaks the molecule into
fragments, commonly a cation and a radical.
– Bonds break to give the most stable cation.
– Stability of the radical is less important.

• Alkanes
– Fragmentation often splits off simple alkyl groups:
• Loss of methyl M+
- 15
• Loss of ethyl M+
- 29
• Loss of propyl M+
- 43
• Loss of butyl M+
- 57
– Branched alkanes tend to fragment forming the
most stable carbocations.

• Mass spectrum of 2-methylpentane

• Alkenes:
– Fragmentation typically forms resonance
stabilized allylic carbocations

• Aromatics:
– Fragment at the benzylic carbon, forming a resonance
stabilized benzylic carbocation (which rearranges to the
tropylium ion)
M+
CH
H
CH Br
H
C
H
H
or

• Alcohols
– Fragment easily resulting in very small or missing
parent ion peak
– May lose hydroxyl radical or water
• M+
- 17 or M+
- 18
– Commonly lose an alkyl group attached to the
carbinol carbon forming an oxonium ion.
• 1o
alcohol usually has prominent peak at m/z = 31
corresponding to H2C=OH+

• MS for 1-propanol
M+
M+
-18
CH3CH2CH2OH
H2C OH

• Amines
– Odd M+
(assuming an odd number of nitrogens are
present)
– α-cleavage dominates forming an iminium ion
CH3CH2 CH2 N
H
CH2 CH2CH2CH3 CH3CH2CH2N CH2
H
m/z =72
iminium ion

86
CH3CH2 CH2 N
H
CH2 CH2CH2CH3
72

• Ethers
– α-cleavage forming oxonium ion
– Loss of alkyl group forming oxonium ion
– Loss of alkyl group forming a carbocation

H O CHCH3
MS of diethylether (CH3CH2OCH2CH3)
CH3CH2O CH2
H O CH2

Aromatics may also have a peak at m/z = 77 for the benzene
ring.
NO2
77
M+
= 123
77

• Aldehydes (RCHO)
– Fragmentation may form acylium ion
– Common fragments:
• M+
- 1 for
• M+
- 29 for
RC O
R (i.e. RCHO - CHO)
RC O

• MS for hydrocinnamaldehyde
M+
= 134
C C C H
H
H
H
H
O
133
105
91
105
91

• Ketones
– Fragmentation leads to formation of acylium ion:
• Loss of R forming
• Loss of R’ forming RC O
R'C O
RCR'
O

• MS for 2-pentanone
CH3CCH2CH2CH3
O
M+
CH3CH2CH2C O
CH3C O

• Esters (RCO2R’)
– Common fragmentation patterns include:
• Loss of OR’
– peak at M+
- OR’
• Loss of R’
– peak at M+
- R’

Frgamentation Patterns
M+
= 136
C
O
O CH3
105
77 105
77

Rule of Thirteen
• The “Rule of Thirteen” can be used to identify
possible molecular formulas for an unknown
hydrocarbon, CnHm.
– Step 1: n = M+
/13 (integer only, use remainder in
step 2)
– Step 2: m = n + remainder from step 1

Rule of Thirteen
• Example: The formula for a hydrocarbon with
M+
=106 can be found:
– Step 1: n = 106/13 = 8 (R = 2)
– Step 2: m = 8 + 2 = 10
– Formula: C8H10

Rule of Thirteen
• If a heteroatom is present,
– Subtract the mass of each heteroatom from the
MW
– Calculate the formula for the corresponding
hydrocarbon
– Add the heteroatoms to the formula

Rule of Thirteen
Example: A compound with a molecular ion
peak at m/z = 102 has a strong peak at 1739 cm-1
in its IR spectrum. Determine its molecular
formula.

References
• Chatwal GR, Anand SK. Instrumental method
of chemical analysis, Himalaya publishing
house.
• Sharma YR. Elementary organic spectroscopy.
• Willard,merritt,dean. Instumental methods of
analysis.
• www.google.com/images
06/13/15 110

Mass spectrometry

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