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Mass spectroscopy
Mass spectroscopy is a quantitative and qualitative analytical technique by
which we can measure the molecular mass and formula of a compound and
the record is known as mass spectra.
Principle/function:
The mass spectrometer is designed to perform four basic functions −
• To vaporize the compound by increasing volatility.
• To generate the ions from the neutral compound in resulting vapor
pressure
• To separate the ions according to their mass to charge ratio (m/z) in
a magnetic field.
• To collect the mass and record.
Theory:
- e- + - e- -
[M] [M] or, [M] [M]
1
M. Kaisarul Islam, Lecturer, Department of Pharmacy, Jagannath University, Dhaka-1100, Bangladesh
2. Fragmentation:
When the molecule has been bombarded by high-energy
electrons to produce ions, the molecule absorbs sufficient
energy and undergo fragmentation.
B+ + neutral
A+
C+ + neutral
D+ + neutral
3. Separation of ions:
The mixture of ions are separated according to the mass charge
ratio in the analyzer and then recorded.
The record is known as the mass spectrum. It is the record of
abundance of each ion against its m/z value.
4. Mass spectrum:
The mass spectrum is a plot of ion current intensity (ion
abundance) versus m/z value.
The most abundant peak will give the tallest peak of the mass
spectra. This peak is known as the base peak and its mass
arbitrarily assigned a value of 100%. The heaviest peak is the
molecular ion peak and its mass will give the mass of the
molecule.
C+
B+
Relative absorbance ↑ D
A+
m/z value →
2
M. Kaisarul Islam, Lecturer, Department of Pharmacy, Jagannath University, Dhaka-1100, Bangladesh
Isotope peak:
Isotopes present in the molecule may generate additional peak. Due to the
occurrence of isotopes we also observe M+1, M+2, M+3 etc peaks. The
relative abundances of these isotopic peaks are proportional to the
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abundance of the isotope in nature (e.g. the natural abundance of C is
1.1% of the 12C atoms. For an ion containing n number of carbon atoms, the
abundance of isotope peak is nX1.1% of the 12C containing peak.
M+1 peaks are made by − 13C, 2H, 15N, 33S
Base peak
M+ peak
Relative abundance ↑
M +1
M+2
m/z ratio →
C6H4D2+.
3
M. Kaisarul Islam, Lecturer, Department of Pharmacy, Jagannath University, Dhaka-1100, Bangladesh
Ionization method:
In ionization method compound are divided into 2 groups −
a. Ionization of volatile materials
b. Ionization of nonvolatile materials
4
M. Kaisarul Islam, Lecturer, Department of Pharmacy, Jagannath University, Dhaka-1100, Bangladesh
M + e = M+ + 2e
+
CH 4 + e → CH 4 • + 2e
+
CH 4 • + CH 4 → CH 5 + + CH 3 •
+
If the sample molecules are volatilized into mixture of ions, CH 5 act
as a strong acid and protonates the sample.
M + CH 5 + → MH + + CH 4
Thus in positive ion CI-spectra, the observed m/z value is one unit greater
than the true molecular weight.
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M. Kaisarul Islam, Lecturer, Department of Pharmacy, Jagannath University, Dhaka-1100, Bangladesh
1. Field desorption:
• Here, the probe tip is replaced by a thin wire on which sharp
needles have been grown.
• The wire is supported between two posts on the probe.
• A solution of small amount of a sample is deposited on the wire.
• In the mass spectrometry the wire is maintained at +8kv and can
be heated and this can cause the discharge of an electron from
the sample into the metal. Thus positive ions (M+) are created.
In this way molecules are thrown into the gas phase as a
positive molecular ion without thermal decomposition.
Cathode slit
+8KV +
wire (+8KV) +
+
Probe +
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M. Kaisarul Islam, Lecturer, Department of Pharmacy, Jagannath University, Dhaka-1100, Bangladesh
Laser desorption →
In this technique, the sample is bombarded with short duration,
intense pulses of laser light.
Efficient and controllable energy transfer to the sample requires
resonant absorption of the molecule at the laser wavelength.
Therefore, lasers emitting either in the UV or IR are employed.
Laser pulses are applied for 1-100ns.
One disadvantage is some thermo labile compounds may be
degraded with the laser beam resulting in a spectrum of
fragment ions. To overcome this problem, a matrix is used and
the technique is known as matrix assisted laser desorption
ionization technique (MALDI).
Matrix Application
Peptides, protein, lipids,
2,5-dihydroxy benzoic acid (DHB)
oligosaccharides
3,5-dimethoxy-4-hydroxy cinnamic
Peptides, protein, glycoproteins
acid (Sinapinic acid)
α-cyano-4-hydroxy cinnamic acid Peptides, protein, lipids,
(CHCA) oligonucleotides
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M. Kaisarul Islam, Lecturer, Department of Pharmacy, Jagannath University, Dhaka-1100, Bangladesh
FAB →
Here the energy is provided by a beam of neutral atom. The
sample is dissolved in a matrix of low volatility. A few µgm of
the sample are dissolved in a few µl of glycerol as matrix and a
beam of fast xenon atoms bombards the solution.
Xe+ + Xe → Xe + Xe+
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M. Kaisarul Islam, Lecturer, Department of Pharmacy, Jagannath University, Dhaka-1100, Bangladesh
252
Cf fission + −
or
Sample
Ni foil (10–3 mm)
Where,
m H 2r 2 r = radius of circular of path in which ion is
= traveling
z 2v H = magnetic field strength
V= potential difference of ion.
9
M. Kaisarul Islam, Lecturer, Department of Pharmacy, Jagannath University, Dhaka-1100,
Bangladesh
where
m 1 t = time of flight
t=L × L = distance in which a ion travel
e 2V m = mass of the ion
e = charge of the ion
V = velocity in which the ion travel
It is quicker than any other mass analyzer and applicable for all
masses.
Quadrupole:
Here four parallel rods arranged symmetrically around an ion flight
path and a direct current and a radio frequency are applied to the
rods resulting an oscillating electrostatic field.
The ions when pass through the region, will acquire an oscillation in
the electrostatic field. The ions of incorrect m/z ratio undergo an
unstable oscillation and strikes one of the rods.
Ions of correct m/z ratio undergo stable oscillation of constant
amplitude and pass through analyzer to reach the recorder.
10
M. Kaisarul Islam, Lecturer, Department of Pharmacy, Jagannath University, Dhaka-1100,
Bangladesh
11
M. Kaisarul Islam, Lecturer, Department of Pharmacy, Jagannath University, Dhaka-1100,
Bangladesh
12
M. Kaisarul Islam, Lecturer, Department of Pharmacy, Jagannath University, Dhaka-1100,
Bangladesh
13
M. Kaisarul Islam, Lecturer, Department of Pharmacy, Jagannath University, Dhaka-1100,
Bangladesh
Example –
Penazitidine A is a heterocyclic compound with a long chain. It has a
methyl group on the side chain but the position was not established by
various technique (NMR & even by 2D NMR). But MS-MS can determine
the position of methyl group on the structure.
The ion at m/z 296 was selected. It was allowed to pass into the
collision chamber where it is subjected to CAD. The mixtures of ions
produced are analyzed in the 2nd mass analyzer. The intense peak at
m/z 182, m/z 210 indicates the position of methyl group at C12 of the
side chain.
Fragmentation patterns:
For most classes of compounds, the mode of fragmentation is
somewhat characteristic. The most common mode of fragmentation
involves the cleavage of one bond. In this process the odd-electron
molecular ion yields an odd-electron neutral fragment (a radical) and
an even electron fragment ion (carbonium type)
14
M. Kaisarul Islam, Lecturer, Department of Pharmacy, Jagannath University, Dhaka-1100,
Bangladesh
+ +
[RCH − CHR ] / •
→ [RCH = CHR / ]• + H 2O
+ +
[RCH − CH 2 − O − C − CH3 ] →
•
[RCH = CH ] 2
•
+ HO − C − CH3
(odd electronmolecularion) (odd electronfragmention) (evenelectronneutralfragment)
CH3-CH-CH3
CH3
0
2 carbonium ion, m/z =43
15
M. Kaisarul Islam, Lecturer, Department of Pharmacy, Jagannath University, Dhaka-1100,
Bangladesh
Alkanes − Molecular ion peak observed at C-C bonds breaks resulting in a homologous
ii)Straight m/z = 58 series of fragmentation products. Primary
chain carbonium ion is formed.
compound + 1. Cleavage of C-1 to C-2 bond result in loss of
[CH 3 − CH 2 − CH 2 − CH 3 ]•
e.g. − Butane a Me-radical and formation of propyl
carbonium ion.
[CH 3 − CH 2 − CH 2 ]+ + [CH 3 ]• (m/z = 43)
43
2. Cleavage of c-2 to c-3 bond result in loss of a
Et-radical and formation of ethyl carbonium
29
ion
15 58 [CH 3 − CH 2 ]+ + [CH 3 − CH 2 ]• (m/z = 29)
3. Cleavage of c-3 to c-4 bond results in loss of
a propyl radical and formation of methyl
11 15 25 30 40 45 50 55 60 carbonium ion
(m/z) → [CH 3 ]+ + [CH 3 − CH 2 − CH 2 ]• (m/z = 15)
Fig: Mass spectrum of Butane
Alkenes − Distinction M+ peak Alkene isomers show nearly identical mass spectra.
E.g. − 1-butene So double bond can’t be located. Also cis & trans
& 2-butene • 1-butene and 2-butene can’t be differentiated.
give molecular peak at
CH2=CH-CH2- m/z =56. +
CH3
[R − CH 2 − CH = CH 2 ] → R + CH2 − CH = CH 2
• • +
+
[CH 2 = CH− CH2 − CH3 ]• → CH3 + CH2 = CH− CH2
• +
(m/z = 41)
+
Alkynes − • Molecular ion peak is
[H − C ≡ C − CH 2 − R] → R • + •
at m/z = 40 +
+ +
[HC ≡ C − CH 3 ]• → HC = C C H 2 ↔ H C = C = CH 2
(Propyne) (m/z = 39)
16
M. Kaisarul Islam, Lecturer, Department of Pharmacy, Jagannath University, Dhaka-1100,
Bangladesh
Aromatic • Show distinct and intense Fragmentation occurs at benzylic position not at
hydrocarbon molecular ion peak. phenolic position.
i) containing
alkyl group
E.g. • Not so intense Fragmentation occurs at benzylic position. Loss of
Tolune • Shows very intense hydrogen gives a strong peak at m/z = 91
molecular ion peak at m/z
CH3 = 92
+ +
CH2
+
CH3
+
C7H7
Benzyl carbonium ion Tropylium ion (m/z = 91)
(m/z = 91)
Ethyl benzene Molecular ion peak gives at Fragmentation occurs at benzylic position. Loss of
m/z = 106 hydrogen gives a strong peak at m/z = 91
CH2-CH3 +
CH2 +
+
CH2-CH3 + CH3
Propyl Molecular ion peak at m/z = Fragmentation occurs at benzylic position. Loss of
benzene 120 hydrogen gives a strong peak at m/z = 91
+ +
CH2
CH2-CH2-CH3 +
CH2-CH2-CH3 + CH2-CH3
Alcohol Intensity of molecular ion • Loss of an alkyl group. C-C bond broken.
•
1º & 2º peak is usually low. CH3 − CH2 − C H 2 + CH2 = OH+ m/z=31
+
3º [CH3 − CH2 − CH2 − CH2 − OH] •
(1 − butanol) •
CH3 − C H 2 + CH3 - CH = OH+ m/z=45
+
[CH3 − CH2 − CH − OH]• •
CH3 C H 3 + (CH3 ) 2 - C = OH+ m/z=45
(2 − butanol)
17
M. Kaisarul Islam, Lecturer, Department of Pharmacy, Jagannath University, Dhaka-1100,
Bangladesh
(CH2)n (CH2)n
Example - (1-butanol)
(CH2)2
CH2
CH2 CH2 + H2O
CH2 CH2
H OH
C
H2
Ex − 1-butanol −
+
H
O
H +
H2C
H2C CH2 + CH2=CH2 + H2O
H2C CH2
m/z = 28
C
H2
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M. Kaisarul Islam, Lecturer, Department of Pharmacy, Jagannath University, Dhaka-1100,
Bangladesh
Ethers − Weak molecular ion peak. • Loss of alkyl group/cleavage of C−C bond to
the α-carbon
α + +
[R − C H 2 OR' ] → CH 2 = O R'+ R •
•
Aliphatic Molecular ion peak is • α-Cleavage: cleavage occurs in one of the two
aldehyde − observed/ weak bonds to the carbonyl group.
O
+
R C H R C O + H *
O
+
R C H H C O + R *
R C H H C O * +R+
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M. Kaisarul Islam, Lecturer, Department of Pharmacy, Jagannath University, Dhaka-1100,
Bangladesh
CH3-CH2-CH2-C-H +
H-C O + CH3CH2CH2 m/z=29
α-Cleavage:
+
O
+
CH3-CH2-CH2-C-H CH3CH2 + CH2=CHO m/z=29
β - Cleavage:
Mclafferty rearrangement:
H +
CH2 +
O +
CH2 CH2 + CH2 CH -OH m/z=44
CH2
CH
CH2
C H 106(M+) m/z=105
105
77 • Loss of CHO group may lead to C6H5+
which show molecular peak at m/z = 77
C6H5+ C6H5C≡O+
Fig: mass spectrum of
Benzaldehyde
Aromatic Intensed molecular ion peak • loss of alkyl radical giving ArCO+ ( α -
ketones − Cleavage)
+
O +
C O +
C R +R*
m/z=105 m/z=77
20
M. Kaisarul Islam, Lecturer, Department of Pharmacy, Jagannath University, Dhaka-1100,
Bangladesh
+
O
+ *
R '- C O + R
R --- C ---R '
2. Mclafferty rearrangement:
+
+
H OH
R 1C H R 1 CH
O + C CH2
R 2C H R 2C H
CR3 R3
CH2
e.g.− +
O
+
2-butanone CH3-C O + CH3CH2
CH3-C-CH2CH3
m/z=43
larger gr. will be predominant
+
O
+
CH3CH2-C O + CH3
CH3--C-CH2CH3
m/z=57
Aromatic Intensed molecular ion peak Loss of OH to form C6H5CO+ ion (m/z = 105)
Carboxylic followed by loss of CO to form the C6H5+ ion
acids − O + +
+
* C O * +
C OH
m/z=105 m/z=77
+ OH *
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M. Kaisarul Islam, Lecturer, Department of Pharmacy, Jagannath University, Dhaka-1100,
Bangladesh
Aromatic Weak molecular ion peak α - Cleavage gives to the formation of C6H5CO+
esters − 105 O + +
C O +
*
E.g. − Methyl C OCH3
benzoate. 77 m/z=105 m/z=77
136 + OCH3*
+
(M )
+
R C-OR R *
+ O C-OR'
'
O + O
*
+
R COR' R + *
C-OR'
+
O
*
+
R-C OR' R C O + *
OR'
22