Transcript Mass spectrometry

1
Mass Spectrometry &
Applications
Ashraf M. Mahmoud, Associate Professor
Contents
I.
Introduction: (Basics of Mass Spectrometry)
II.
1.
2.
3.
4.
5.
Ionization step:
Electron Ionization
Chemical Ionization
Photoionization
Matrix-assisted Laser Desorption/Ionization
Electrospray Ionization
III.
1.
2.
3.
4.
Mass Analysis (Separation):
Magnetic Sector
Quadrupole
Ion Trap
Time-of-flight
IV. Molecular fragmentation pattern
V. Major Applications of Mass Spectrometry
Theory of Mass Spectrometry
Schematic of Mass Spectrometry
Ionizer
↓
Mass-to-charge ratio Analyzer
↓
Detector
channeltron, electromultiplier and microchannel plates.
Theory of Mass Spectrometry
2
 In a mass spectrometer, molecules in the gaseous state under low pressure
(high vacuum) are bombarded with a beam of high energy electrons (70 eV)
 This bombardment can first dislodge one of the electrons of the molecule and
produce a positively charged ion called “The molecular ion”→ ionization step
M
+
Molecule
e’
high-energy
→
M+•
+
2 e’
molecular ion
 The molecular ion contains also an odd number of electrons, thus it is a Free
radical, or generally “Radical Cation”
 An electron beam with an energy of about 70 eV not only produces molecular
ions but also give to the molecular ions considerable amount of surplus energy
which is quite sufficient to break any covalent bond in the molecular ion (4-7 eV)
Thus soon after they are formed, most molecular ions undergo fragmentation.
 Fragmentation can take place in a variety of ways depending on the nature of
the molecular ion.
Ionization step
Different types of systems are commonly used to ionize the
Molecule:
1. Electron Ionization
2. Chemical Ionization
3. Photoionization
4. Matrix-assisted Laser Desorption/Ionization
5. Electrospray Ionization
Ion separator systems
Different types of systems are commonly used to separate
ions on the basis of their m/z ratio → Ion separation step
such as:
1. Magnetic sector instrument
Advantages :
a. High accuracy
c. Resolve ions to a level of 0.0001 amu or more
Disadvantage :
a. Its Lower sensitivity
b. Narrow range of kinetic energy
2. Quadrupole instrument
It uses two electric fields applied at right angles to each other, rather
than a magnetic field, to separate ions according to their m/z ratios,
one of them is oscillating to create a resonance frequency for each
m/z value. The ions which resonate at the frequency of the
quadrupole are able to pass through it and be detected
Ion separator systems (cont.)
Advantage :
a. Its higher sensitivity b. Wider range of kinetic energy
c. More cheaper than magnetic sector
Disadvantages :
a. Low accuracy
b. It can not resolve ions not more than 0.1 amu
3. Ion trap instrument
4. Time-of-flight instrument
 MS can give us highly useful information about the structure of a
complex molecule.
 MS, then sorts the produced cations on the basis of their m/z ratios.
 Since for all the practical purposes, the charge on all of the ions = +1,
thus the sorting of them actually on the basis of their masses.
Diagram for Mass spectrometer
electrons generator
that give the high
energy electrons
beam
Ionization region
Heater to generate the
gas beam of molecules
Detector
Mass Spectrum
Mass Spectrum:
1. The mass spectrum—is a graph of relative abundance of each
fragment (% Intensity) plotted against its m/z value.
2. Because the charge (z) = +1 for all fragments that reach the collector
plate so the m/z value express molecular mass (m) of fragments.
3. Remember that only positively charged species reach the collector.
4. The mass spectrum is usually published either as a bar graph or in
table form
Note that:
1. Peak with the highest m/z value in the spectrum is due to the
fragment that results when an electron is knocked out of a molecule
of the injected sample. i.e. represents the molecular ion (M)+ of the
studied compound.
Thus m/z value of molecular ion gives the molecular mass of
compound.
Molecular, Base & Fragment ion peaks
2. Since it is not known what bond loses the electron, the molecular
ion is put in brackets and the positive charge and unpaired electron
are assigned to the entire structure.
3. Peaks with smaller m/z values—called fragment ion peaks—
represent positively charged fragments of the molecule.
4. “Base peak” is the peak with the greatest intensity, due to its
having the greatest relative abundance. The base peak is assigned
a relative intensity of 100%, and the relative intensity of each of the
other peaks is reported as a percentage of the base peak.
5. The mass spectrum gives us structural information about the
compound because the m/z values and the relative abundances of
the fragments depend on the strength of the molecular ion’s bonds
and the stability of the fragments.
Example
Mass spectrum of pentane, shown
as a bar graph and in tabular form.
Base peak represents fragment
that appears in greatest
abundance. Value of molecular ion
gives molecular mass of the
compound.
Note that:
The way by which the
molecular ion to be
fragmented depends
on the strength of its
bonds and stability of
the fragments.
Note that:
1- In some cases the base peak is the molecular ion peak, but in
most cases it is different from that of the molecular ion (according
to the relative stability of ions during fragmentation).
2- Peaks are commonly observed at m/z values one and two units
less than the m/z values of the carbocations because the
carbocations can undergo further fragmentation by losing one or
two hydrogen atoms.
3- Small peak that occurs at m/z 73 (0.52%) is known as M+1
peak to indicate that “It is one mass unit greater than the
molecular ion(M+.),
The M+1 peak appear because most of elements have more than
one naturally occurring isotope.
Relative abundances of different isotopes
Principal stable isotops of common elements:
From the table :
•For C, H, N and Si the principal heavier isotope is M+1.
•For O, Si, S, Cl and Br , the principal heavier isotope is the M+2
•For F and I, there is no heavier isotopes (not affect M+1 or M+2)
Some guides to determine molecular
formula of organic compounds:
Nitrogen rule: If molecular ion (M+) is even number, compound must contain an
even number of N atoms (zero is considered as even number)
• Number of Carbon atoms: Relative abundance of M+1 peak can be used for
determination of number of carbons assuming that silicon and large number of
nitrogens (more than 3 N) are not present.
No of Carbons = Relative abundance of M+1/ 1.10
• Relative abundance of M+2 peak indicates the presence (or absence) of Oxygen
(0.2), Silicon (3.35),Sulfur (4.4), Chlorine (32.5) & Bromine (98.0)
• Molecular formula after this can be established by adding the suitable number
of hydrogens and oxygens if necessary.
• The index of hydrogen deficiency (Due to double and triple bonds and
cyclization) can be calculated for the compounds containing C, H, N, O, S &
halogens from the formula:
• Index = Carbons - Hydrogens/2 – Halogens/2 + Nitrogens/2 +1
•Divalent atoms like O and S are not counted in formula. The index mainly used
to indicate the number of double bonds.
Examples:
Determine the molecular formula for the following compounds
M/Z
%
12
Note
M/Z
%
2.6
16
13
8.6
14
Note
M/Z
%
0.9
14
17
21.1
17.1
18
100
15
85.6
19
16
100
M+
20
17
1.15
M+1
Note
M/Z
%
Note
17.2
73
0.56
M+1
15
100
72
18.56
M+
M+
19
2.0
71
4.32
0.06
M+1
31
10.4
57
11.20
0.2
M+2
33
89.4
43
100.0
34
95.4
M+
29
26.65
35
1.1
M+1
15
4.22
GC-MS
1. Constructed By interfacing the GC to the MS by using jet separator
2. Different types of ionization were used:
a. Electron impact
b. Positive ion chemical ionization (reagent gas is used, methan,
isobutane, or ammonia; gas interacts with electrons to produce
positive ions which can either associate with the analyte or transfer a
proton to the analyte
c. Negative ion chemical ionization
LC-MS
1. Constructed By interfacing the LC to the MS by using different types of
interfaces
a. Particle beam
b. Thermospray (reagent gas is used, methan, isobutane, or ammonia;
gas interacts with electrons to produce positive ions which can either
associate with the analyte or transfer a proton to the analyte
c. Fast atom bombardment
d. Electrospray ionization
e. Atmospheric pressure ionization
Major Applications of Mass Spectrometry
1. MS provides a highly specific method for determining or confirming
the structure or the identity of drugs and raw materials.
2. MS in conjunction with either gas chromatography (GC-MS) or liquid
chromatography (LC-MS) provides a method for characterizing the
impurities.
3. GC-MS and LC-MS provides a highly sensitive and specific method
for determining drugs and their metabolites in biological fluids and
tissues.
4. MS has become an important tool in proteomics, which is currently
the major tool in drug discovery
5. MS using electrospray ionization and time of flight separation will be
of major use in quality control of therapeutic antibodies and protein.
6. It is the best method for getting rapid identification of trace
impurities.
Limitations of Mass Spectroscopy
1. It is not currently used in routine quality control due to complexity of
instrumentation
2. Expensive instrumentation
3. requires a high degree of skill
4. Regular maintenance
Examples
The IR and mass
spectra for a compound
is shown in the
following Figure,
Identify the compound
If you know that M+ at
m/e 100 (20%), M+1
at 101(1.32%) and
M+2 at 102 (0.04%) .
Operation of Mass Spectrometry
•This operation begins by producing the gas beam of molecules,
bombardment with a beam of high energy electrons and accelerating
the ions beam through its pass through the curved tube.
•This curved tube (Analyzer tube) passes through a variable magnetic
field and the magnetic field exerts an influence on the moving ions
deflecting the positively charged fragments in a curved path.
•At a given magnetic field strength, the degree to which the path is
curved depends on the mass-to-charge ratio (m/z) of the fragment.
•Path of a fragment with a smaller m/z value will bend more than that of
a heavier fragment. In this way, the particles with the same m/z values
can be separated from all the others.
•If a fragment’s path matches the curvature of analyzer tube, the
fragment will pass through the tube and out the ion exit slit (These ions
are said to be in register).
Operation of Mass Spectrometry (cont.)
•A collector records the relative number of fragments with a particular
m/z passing through the slit. The more stable the fragment, the more
likely it will make it to the collector.
•Strength of magnetic field is gradually increased, so fragments with
progressively larger m/z values are guided through tube and out the
exit slit.
Note that:
•Actual sorting of ions takes place first in the gradually increased
magnetic field
•Ion-sorting can also be done with “Electrical Focusing”, in this
technique the magnetic field is held constant and the accelerating
voltage is varied. It gives the same sorting results.
•In the high-resolution mass spectrometers both techniques are used
to increase the system sensitivity.
Example:
If the molecule under bombardment is a molecule of ammonia, the
following reaction will take place:
H:N:H
H
H:N:H
H
+
H:N:H
+
H
Molecular ion
of ammonia
e'
H:N:H
+
H
H:N:
2 e'
+
H
:N: + H
Molecular fragmentation pattern
1. Homolytic and heterolytic α-cleavage
- under electron impact ionization condition, the analyte developes a +ve
charge through loss of one electron.
- If there is an electronegative atom such as oxygen, sulphur or nitrogen, this
+ve charge will be located on the electronegative atom.
- If electronegative atom is absent, the charge is more difficult to locate with
certainity.
a. Homolytic α-cleavage
- is promoted by the presence of hetero-atom
- it often gives rise to the most abundant ion peak (base peak)
- The hetero-atom becomes +vely charged
- it favors the loss of the largest possible radical
- This type of fragmentation dominates the mass spectra of many drugs (major
pathway)
b. Heterolytic α-cleavage
- is promoted by the presence of hetero-atom
- The hetero-atom is not the+vely charged ion
- it does not favor the loss of the largest possible radical
- This type of fragmentation is a minor pathway
2. Cleavage with proton transfer
- is also common pathway in the mass spectra
Molecular fragmentation pattern (cont.)
3. McLafferty rearrangement
- is not particularly common pathway in mass spectra of drugs
- occurs in carboxylic acids, esters, ketones and amides which
have side chain of at least 3 carbon atom
- associated with elimination of small, stable, neutral molecules such
as carbon monoxide, olefines, water, ammonia,
- it characterise the long chain lipid molecules
-To undergo a McLafferty rearrangement, a molecule must possess an
appropriately located heteroatom , a p-system (usually a double
bond), and an abstractable hydrogen atom g to the C = O system.
Examples
The IR and mass
spectra for a compound
is shown in the
following Figure,
Identify the compound
If you know that M+ at
m/e 162 (100%), M+1
at 163(6.62%) and
M+2 at 164 (98.0%) .
Examples
The IR and mass
spectra for a
compound is shown in
the following Figure,
Identify the compound
If you know that M+
at m/e 72 (50%),
M+1 at 73(2.3%) and
M+2 at 74 (0.1%) .
Ashraf M Mahmoud