mass spectroscopy
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Mass Spectrometry
1
?Why it is use Mass Spectroscopy ?
2
Introduction to Mass Spectrometry
Sample introduction
Ionization
Minimize collisions,
interferences
Separatemasses
Count ions
Collect results
3
PrincipleIt is also called as positive ion spectra or line spectra Sample is bombarded with the high electron beam produce the positive ions.
They travel in straight path
When a magnetic field or electric field is applied then travels in curved path
The fragments of different masses are separated based on the radius of curvature. m/e α r2
4
How does a mass spectrometer work?
Sample PlateTargetHPLCGCSolids probe
MALDIESIIonSprayFABEI/CI
SFADFAQuadrupoleFTMS
Faraday cup .Electron Mult.Photomultiplier
5
V i d e o
6
Mass Spectrometry Needs
Ionization-How the protein is injected in to the MS machine
Separation-Mass and Charge is determined
Activation-Protein are broken into smaller fragments (peptides/AAs)
Mass Determination- m/z ratios are determined for the ionized protein fragments/peptides
?7
FRAGMENTATIONThe process of Breaking Molecules /ions into
fragments is known as fragmentation.This can be seen in the form of peaks in mass spectra Methanol can be divided in to 4fragmentse.g. CH3OH CH3OH +e¯⁺
CH3OH CH3 + OH¯⁺
CH3OH CH2OH + H¯⁺
CH3OH CHO + H2¯⁺
.
5 10 15 20 25 30 35
120
100
80
60
40
20
0
CHO⁺
CH3OH⁺
CH3⁺
CH2OH⁺
m/e
inte
nsi
ty
8
Fragmentation rules in MS
9
1. Intensity of MM.+.+ is Larger for linear chainLarger for linear chain than for branched compound
2. Intensity of MM.+.+ decreasedecrease with IncreasingIncreasing M.W.M.W. (fatty acid is an exception)
3. Cleavage is favored at branchingfavored at branching
4.4. Aromatic Rings, Double bond, Cyclic structures stabilizeAromatic Rings, Double bond, Cyclic structures stabilize MM.+.+
5. 5. Double bond favor Allylic CleavageDouble bond favor Allylic Cleavage
6. Saturated Rings lose a Alkyl Chain (case of branching)
7. 7. Aromatic Compounds Cleave in b Aromatic Compounds Cleave in b Resonance Stabilized TropyliumTropylium
8. 8. C-C C-C Next to HeteroatomNext to Heteroatom cleave leaving the charge on the charge on the HeteroatomHeteroatom
9. 9. Cleavage of small neutral molecules (COCleavage of small neutral molecules (CO22, CO, olefins, H, CO, olefins, H22O ….).O ….).
Result often from rearrangement - McLafferty rearrangement Result often from rearrangement - McLafferty rearrangement 9
General rules of Fragmentation
1.Hydrocarbons•Hydrocarbons give clusters of peaks.
•Molecular ion peaks of very low abundance are observed for linear hydrocarbons.•For branched hydrocarbons give a low intensity at M+.•Intensity of (CnH2n+1) peaks decreases with increasing mass.
10
2.Cleavage at Branched carbon
C > C
H
> C
H
H
>H
C
H
H
tert. sec.primary methyl
Cleavage at branched carbon is favored due to higher stability at tertiary carbocation.
General rules of Fragmentation
11
+
cleavage at 6-1
cleavage at 6-3
cleavage at 6-2
C H
C4H9
C3H7
C H
CH3
C4H9
+
+
C H
CH3
C3H7
+
(F1)
(F2)
(F3)
H3C CH2 CH2 C
CH3
H
CH2 CH2 CH2 CH31 2 3 4 5 6 7 8
Eg.
Produces thre secondary cations, the most favored fragments at C-4 of
4- methyl octane.Note that C4 is common for fragments (F1)(F2) And (F3). 12
3.Rule of cleavage
X C1C2 R X CH
a b
Most important rule covers 70% of mass fragmentation.
Cleavage favored at bond leaving positive charge on C1.
General rules of Fragmentation
13
H3C CH2 O CH2 CH3
H3C CH2 O CH2 CH3
CH2 O CH2
m/e = M-15
1.
H3C
2.
H3C CH2 N CH2
CH2 CH2 CH3
NC2H5
C3H7
H2C
m-57m-29
NC2H5
H2C
H2C
NCH2
C3H7
m-15
CH2
tert.amine
B1B2B3
e.g.: A) (x) = O, N, S.
14
3.
CH2 S CH2 CH2 CH3
SH2C
CH2
SH2C
C3H7M-71
M-29
B2 B1
B1B2
R
CH2CH2
+
+
m/e = ( M-R )Stablebenzylic cation
+
m/e = 91
Tropylium cationm/e = 65
cyclopentadienylcation
+
b)
b) Benzylic clevage
-(x)- =
16
Very common fragment for ester
M-31 = methyl esterM-45 = ethyl ester
C. Allylic Cleavage
H2C
R
m/e = M-R stable allyliz cation
CH3H3C
O
R CH3
O+
R C O+CO CH3
m/e = M-R m/e = M-15
Simarly for x= N & S
i)
ii)
CR OCH3
O
CR O+
m/e = M-31
+
17
4 Rule of elimination of small neutral molecule
C
H
C
OH
C C
+
+ H2O
m/e M - 18
A) - EliminationThe high temperature and high vacuum are quite favourable for elimination reaction
and hencei)Loss of water (H2O) for alcohols (M-18) is a prominent fragment.Tertiary alcohols lose the water so fast that in many cases M.I. Peak is absent.
General rules of Fragmentation
18
C C
NH
C C + NH2
M - 46
C2H5
C2H5
ii)Loss of Ammonia (NH3)(M-17) for primary amines and primary and secondary alkyl ammonia derivatives For
C
H
C
NH2
C C +
M - 17
NH3
19
iii)Elimination at Hydrogen sulphide (H2S)[M-34] confirms thiols (mercaptons)
C
H
C
SH
C C + H2S
M - 34
iv)Elimination of Hydrogen cyanide (HCN)[M-27] confirms nitriles.
C
H
C
CN
C C + HCN
M - 27
20
v)Elimination of Hydrogen halide(HX),
Common for tertiary halides.
C
H
C
X
C C
m/e = M - HX X = F, Cl, Br, I
21
5.Rule – retro Diel’s Alder reaction
High temperature high vacuum highly favorable for(DA) common for all these six membered cyclic mono olefins.
+
O
O
O + O
O
O
diene dienophile
General rules of Fragmentation
22
MCLAFFERTY REARRANGEMENT:-
Rearrangement ions are fragments, they are formed due to the result of intermolecular atomic rearrangement during fragmentationTo undergo this rearrangement the molecule must posses heteroatom, one double bond and hydrogen atom
McLaffertyMcLafferty
x
CH2
CH2
H
CH2
O
CY
Y Y H, R, OH, NR2 H, R, OH, NR2
Ion Stabilized Ion Stabilized by resonanceby resonance
x
CH2
CH2
H
CH2
O
CY
- CH- CH22=CH=CH22
x
CH2
O
CY
H
CH2+
O+
CY
H
x
CH2+
O
C+
Y
H
23
It is used for determination of molecular mass of compounds and its elemental composition
Molecules having odd mass number contain odd number of nitrogen atoms.
Molecules having even mass number contain even no of nitrogen atoms.
NITROGEN RULE:-
CH3
CH3 CH3
H
MW = 59 MW = 59 (odd)(odd)
MW = 58 MW = 58 (even)(even)
Ionisation Ionisation [M+H][M+H]
[M+H][M+H]
MW = 60MW = 60
MW = 59MW = 59
CH3
N
CH3 CH3
24
Nitrogen:Odd number of N = odd MW
CH3CNM+ = 41
SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced Industrial Science and Technology, 11/2/09)
25
Problems and General pattern for individual Families
26
Fragmentation Patterns
Alkanes:Fragmentation often splits off simple alkyl groups:
Loss of methyl M+ - 15Loss of ethyl M+ - 29Loss of propyl M+ - 43Loss of butyl M+ - 57
Branched alkanes tend to fragment forming the most stable carbocations.
27
Fragmentation Patterns
Mass spectrum of 2-methylpentane
CH2
CH3 CH3
CH2
CH2
CH2+
CH3 CH3
CH2+
CH2
CH2
CH3 CH3
CH2
CH3 CH3
CH2+
CH2
CH
CH3 CH3
Aklenes (olefins)
CH2
CH3 CH3
CH2
CH3 CH3
m/z 69 m/z 67 m/z 93
Fragmentation Patterns
Fragmentation Patterns
Hydroxy compounds:Hydroxy compounds:
R2 C
R3
R1
O Hx
Loss of largest groupLoss of largest group
- R3
R2
CR1
O+
HR2
C+
R1O H
If RIf R11=H m/e 45, 59, 73 …=H m/e 45, 59, 73 …
If RIf R11=alkyl m/e 59, 73, 87 …=alkyl m/e 59, 73, 87 …
x
OH
CHR
H
CHR
CHR
CHR
OH+
CHRCHR
CHR
CHR
H
CHR+
CHR
CHR
CHR CHR+
CHR
CHR
CHR
x
OH
CHR
H
CHR
CHR
CHR
CHR
CHR
M – (HM – (H22O) – (C1=C2) AlkeneO) – (C1=C2) Alkene
- H2O
- CHR=CHR
M – (HM – (H22O)O)
– – (H(H22O)O)
Fragmentation Patterns
Fragmentation Patterns
Fragmentation Patterns
Aromatics may also have a peak at m/z = 77 for the benzene ring.
NO2
77M+ = 123
77
Fragmentation Patterns
AlcoholsFragment easily resulting in very small or missing
parent ion peakMay lose hydroxyl radical or water
M+ - 17 or M+ - 18Commonly 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+
Fragmentation Patterns
MS for 1-propanol
M+M+-18
CH3CH2CH2OH
H2C OH
SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced Industrial Science and Technology, 11/28/09)
Fragmentation Patterns
Ethers -cleavage forming oxonium ion
Loss of alkyl group forming oxonium ion
Loss of alkyl group forming a carbocation
Fragmentation Patterns
Aldehydes (RCHO) Fragmentation may form acylium
ion
Common fragments:
M+ - 1 for M+ - 29 for
RC O
R (i.e. RCHO - CHO)
RC O
Fragmentation Patterns
Ketones Fragmentation leads to formation
of acylium ion:
Loss of R forming
Loss of R’ forming
RC O
R'C O
RCR'
O
Fragmentation Patterns
MS for 2-pentanoneCH3CCH2CH2CH3
O
M+
CH3CH2CH2C O
CH3C O
SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced Industrial Science and Technology, 11/28/09)
Fragmentation Patterns
Esters (RCO2R’) Common fragmentation patterns
include: Loss of OR’
peak at M+ - OR’
Loss of R’ peak at M+ - R’
Fragmentation Patterns
M+ = 136
C
O
O CH3
105
77 105
77
SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced Industrial Science and Technology, 11/28/09)
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Any
Q.Thank You
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