Spectroscopic Techniques

NMR tells us about the carbon skeleton of the

molecule
IR tells about the types of the bonds (functional
groups) in a molecule.
UV tells us about the conjugation
(chromophores)
What shape does a molecule have?
X-ray answers this question better than any
other method
X-ray gives bond lengths and angles
Why bother about spectroscopy when X
ray is there? y
Co-enzyme of a methanotropic bacteria
Structure solved in 1979
1. You need a crystalline solid
2 It d ifi kill C t ll h !
Structure solved in 1979
2. It needs specific skills Crystallographer!
3. long time for collection and interpretation of data.
Why do we need to know about mass spectrometry?
Mass spectrometry Mass spectrometry
How is it different from IR, UV or NMR spectroscopy
What we measure is not energy but mass of a molecule or
its fragment
It is a plot of abundance Vs mass to charge (m/z).
What is it used for?
Mass spectrometry weighs the molecule.
d f It is used for
1. determining masses of particles
2. Identifying a known compound by comparing with
i ti t l d t existing spectral data
3. determining isotopic elemental composition of a sample,
4. Identification & structure elucidation of a new compound
by observing its molecular ion & fragmentation pattern by observing its molecular ion & fragmentation pattern
5. quantifying the amount of a compound in a sample
Mass Spectrometry Mass Spectrometry
Molecular weight can be obtained from
ll l a very small sample.
A beam of high-energy electrons
b k h l l breaks the molecule apart.
Destructive technique, the sample
b d cannot be recovered.
The masses of the fragments and their
l b d l f relative abundance reveal information
about the structure of the molecule.
4
How does it work
Mass spectrometer has three
essential components
1 Something to volatalize and
Magnetic sector
mass spec.
1. Something to volatalize and
ionize the compound into
beam of charged particles
Electron impact or chemical Electron impact or chemical
ionization an ion source
2. Something that separates
th s i ns b m ss/ch r these ions by mass/charge
ratio - analyzer
3. Something that could
d t t th i detect these ions. -
detector
Main principle: Lighter ions get deflected by the Main principle: Lighter ions get deflected by the
magnetic force more than heavier ions (based on Newtons
second law of motion, F = ma).
Also ions with more charges gets deflected more
Mass Spectrometer
Principle : Ions can be deflected by magnetic field.F = ma
1.Ionization
M ti fi ld b
2.Acceleration
Magnetic field can be
varied to allow all the
streams to pass
through to detector
3.Deflection
4.Detection
http://www.chemguide.co.uk/analysis/masspec/howitworks.html#top,Dr.JimClark
Stages
Sample is ionized (ionization methods, will be discussed) Sample is ionized (ionization methods, will be discussed)
The ions are accelerated so that they all have same kinetic
energy
Th i th d fl t d b ti fi ld di t The ions are then deflected by a magnetic field according to
their masses. The lighter they are, the more they are deflected.
The amount of deflection also depends on the number of positive
h h Th h h d h charges on the ion. The more the ion is charged, the more it gets
deflected. Analyzer separates them on the basis of their mass to
charge ratio (ion separation methods, will be discussed)
The detector detects the beam of ions passing though.
The magnetic field can be varied to cover the entire mass range.
The mass of each ion being detected is related to the size of the The mass of each ion being detected is related to the size of the
magnetic field used to bring it on to the detector.
Mass spectrum is recorded as a stick diagram showing the
l ti t d d b i s f i m ss/ h ti relative current produced by ions of varying mass/charge ratio.
i.e. relative abundance Vs. m/z
Radical Cation Formation Radical Cation Formation
When a molecule loses one electron, it then
has a positive charge and one unpaired has a positive charge and one unpaired
electron. This ion is therefore called a
radical cation.
9
Electron Impact Ionization Electron Impact Ionization
H C
H
C
H
H H C
H
C
H
H
H H
H C
H
C
H
+
H
H H
H C
H
H
C
H
H
H
+
Other fragments can be formed when CC or CH bonds are broken
during ionization. Only the positive fragments can be detected in MS.
10
The Mass Spectrum
In the spectrum, the tallest peak is called the
base peak and it is assigned an abundance of
100% The % abundance of all other peaks are 100%. The % abundance of all other peaks are
given relative to the base peak.
The molecular ion (M
+
) corresponds to the mass of
the original molecule the original molecule.
Example
Resolution
In order to separate two
adjacent peaks of 1 mass unit (Da)
difference the valley between d fference the valley between
them shouldnt be more than
10% of the highest peak. This
degree of resolution is called
unit resolution and can go unit resolution and can go
upto 3000 Da on standard
Instrument. = LRMS.
R=3000/30002999=3000
High resolution
Mass = 250
g
R = 250.1933/ (250.1933-250.1807) = 19857
This is because atomic masses are non integers (except
12
C = 12.00000 by definition)
(R can go upto 100000)
Only HRMS can be used to determine atomic composition of an ion with accuracy Only HRMS can be used to determine atomic composition of an ion with accuracy
Only HRMS can be used to determine
Bee alarm pheromone
Only HRMS can be used to determine
atomic composition accurately
MWCalcdObsError(ppm)
C
6
H
10
O
2
114.068075114.1039358
6 10 2
C
6
H
14
N
2
114.115693114.1039118
C
7
H
14
O 114.104457114.10395
C
6
H
18
114.140844114.1039369
High Resolution MS High Resolution MS
Masses measured to 1 part in million. R 20000-1 lac
A molecule with mass of 44 could be C
3
H
8
C
2
H
4
O CO
2
A molecule with mass of 44 could be C
3
H
8
, C
2
H
4
O, CO
2
,
or CN
2
H
4
.
Using a mass with more significant figures would help
identify the correct formula. identify the correct formula.
For example, lets say the compound we are looking for
has mass of 44.029, pick the correct structure from
the table:
15
Gas ChromatographyMass
Spectrometry (GCMS)
The gas chromatograph column separates the
mixture into its components. p
The mass spectrometer scans mass spectra of
the components as they leave the column.
Typical mass spectrometer Typical mass spectrometer
Lets talk about the various ionization methods first
Ionization methods
Gas phase ionization methods (MW < 1000)
1. Electron Impact ionization (EI) . Electron Impact on zat on (EI)
2. Chemical ionization (CI)
Desorption ionization methods
1. Field desorption ionization (FD)
2 F t t b b d t (FAB) 2. Fast atom bombardment (FAB)
3. Plasma desorption ionization
4 Laser desorption ionization (MALDI)
Evaporative ionization
1 Th m s m ss s t m t
4. Laser desorption ionization (MALDI)
1. Thermospray mass spectrometry
2. Electrospray mass spectrometry (ESI)
Electron Impact Ionization (EI method)
Widely used method.
Vapor phase sample molecules are bombarded with high energy (70
eV) electrons, which eject an electron from sample to create a
radical cation.
Since IP of organic molecule = 15 eV, the excess ~50 eV energy g , gy
is dissipated in breaking other bonds (3-10 eV).
The breaking of bonds is highly extensive and critically, highly
reproducible and is characteristic of a particular compound reproducible and is characteristic of a particular compound
fragmentation pattern. The fragmentation process is also predictable.
Advantages : Major libraries of 390000 compounds searched by
t l ith i EI D t t hi i li bl f computer algorithms are in EI. Data matching is reliable proof.
Coupled with a GC it becomes a powerful tool for structure elucidation
Disadvantage: Sometimes molecular ion peak is not seen. Reduce Voltage.
Chemical Ionization (CI method)
Soft ionization technique to enable molecular ion peak
Sample molecules in vapor phase are not bombarded with high energy Sample molecules in vapor phase are not bombarded with high energy
electrons
Reagent gas (usually methane, isobutane and ammonia are used) is
introduced into the source and ionized introduced into the source and ionized.
Sample molecules collide with ionized gas (CH
5
+
, C
4
H
9
+
etc.) in the
relatively high pressure CI source and undergo secondary ionization by
proton transfer giving [M+1]
+
by electrophilic addition producing proton transfer giving [M+1] , by electrophilic addition producing
[M+15]
+
, [M+24]
+
, [M+43]
+
, or [M+18]
+
(with NH
4
)
+
ions, or rarely by
charge exchange giving M
+
ion. It sometime shows [M-1]+ by hydride
abstraction. Less energy very less fragmentation, abstraction. Less energy very less fragmentation,
Advantage: stable molecular ions enable molecular weight determination
Disadvantage : Not useful for peak matching and structure elucidation
EI Vs CI
Desorption ionization methods
Sample molecules are emitted directly from a condensed
phase into vapor phase as ions
U d f l l til i i d Used for large, non-volatile or ionic compounds
Very less fragmentation
Provides molecular weight or exact mass g
Desorption ionization methods
1. Field desorption ionization (FD) sample is applied to metal
i ( d ) d i hi h l di emitter (anode) and in a very high voltage gradient
2. Fast atom bombardment (FAB)
Uses high energy Xenon or Argon atoms (6-10 Kev) to g gy g ( )
bombard samples in glycerol as a matrix controlled
fragmentation- useful for proteins, carbohydrates etc.
1 Plasma desorption ionization
252
Cf used with TOF 1. Plasma desorption ionization
252
Cf used with TOF
2. Laser desorption ionization (MALDI)- a pulsed laser beam
is used with nicotinic acid or sinapinic acid as a matrix
Comparison
Evaporative ionization
Two methods in which the ions/ compounds in solution Two methods in which the ions/ compounds in solution
have their solvent molecules stripped by evaporation
with simultaneous ionization leaving behind the ions w th s multaneous on zat on leav ng beh nd the ons
for analysis. Coupled with LC.
1. Thermospray mass spectrometry - outdated
2. Electrospray mass spectrometry (ESI or API)
useful for proteins, peptides, multi charged species
Comparison of various ionization methods p
Now, lets talk about the various type of analyzers.
Analyzers Analyzers
Heart of the spectrometer p
There are several types with different
characteristics
1 M i S M S 1. Magnetic Sector Mass Spectrometers
2. Quadrupole Mass Spectrometers
3 I n T p M ss Sp t m t s 3. Ion Trap Mass Spectrometers
4. Time of Flight Mass Spectrometers
5 Fourier Transform Mass Spectrometers 5. Fourier Transform Mass Spectrometers
Tandem Mass Spectrometry Tandem Mass Spectrometry
Magnetic sector
MS were the first commercially available instruments and remained important
Separation is on the basis of momentum, each ion acquires same kinetic energy
The magnetic field is scanned to bring the ions sequentially into focus as per m/z
Single focusing
Combined Electrostatic-Magnetic sector
Very high resolution. Allows measurements of exact masses and molecular formulae
Double focusing
Quadrupole mass analyzers
Much smaller and cheaper
Consists of 4 cylindrical rods (10-20 Cms)
A constant DC voltage modified by a Radio Frequency (RF) is applied g y q y ( ) pp
As ions enter at one end only one m/z ion will pass through, at a
certain combination of DC and modified RF voltage
They are nothing but Tunable mass filters They are nothing but Tunable mass filters
Thus entire mass range can be scanned in much < a second
high sensitivity as no slits
Less mass range 5000
And resolution
Ion Trap Analyzer
Unlike Quadrupole, it can TRAP ions for relatively long period
The ions can be sequentially ejected to the detector to record
spectrum mass-selective instability
Consists of 3 electrodes a ring and two cap electrodes
Ring electrode operates on a sinusoidal RF
The endcap electrode operates either at ground potential/ DC / AC The endcap electrode operates either at ground potential/ DC / AC
First, all the ions above certain cut-off are trapped then the RF
voltage is raised leading to sequential controlled ejection of ions
There are
various
Modes and
Possibilities
For designing
specific
experiments
Using this Using this
analyzer
Tandem MS-MS
Time of Flight (TOF) Concept Time of Flight (TOF) Concept
A packet of stationary ions is A packet of stationary ions is
accelerated to a defined kinetic energy
and the time required to move through a q g
fixed distance is measured
First TOF design published in 1946 by W.E. Stephens
Detector
TOF
The ions are accelerated through a potential and allowed to
drift through a field-free region (a tube = 0.5 to several
meters) to a detector.
Assuming that all the ions arriving at the start of the drift
tube have the same energy, the ions with different mass will
have different velocities.
The time ions arrive at the detector is measured and related
to the m/z ratio.
Unlimited mass range useful for very large biomolecules Unlimited mass range useful for very large biomolecules
Instrument is not scanning, no tunneling, no trapping of ions
No slits, filters, therefore excellent sensitivity
Need to produce the ions at accurately known start time &
position, so use of pulse ionization technique such as plasma
or MALDI is a must
Due to variation in energies of ions, the resolution <20000
Needs very complex and fast electronics for detection
Fourier Transform (FT)
Not very commonly used due to cost
In a FTMS, ions are held in a cell with an electric In a FTMS, ions are held in a cell with an electric
trapping potential within a strong magnetic field. An RF
pulse is applied to generate the FT mass spectrum.
Within the cell each ion orbits in a direction Within the cell, each ion orbits in a direction
perpendicular to the magnetic field with a frequency
proportional to ions m/z. An applied RF pulse brings all
l d l f l l cycloidal frequencies into resonance simultaneously to
yield an interferrogram, which is a time domain
spectrum, which is Fourier transformed into a p
frequency domain spectrum, which then yields a MS.
Needs powerful magnet
V t hi h s l ti n m ss n is Very accurate, high resolution, mass range is
proportional to magnetic field strength
Comparison of various analyzers Comparison of various analyzers
Lowres.,fastelectronics
Tandem Mass Spectrometry (MS-MS) Tandem Mass Spectrometry (MS MS)
In MS-MS (MS squared), a parent ion from the initial
fragmentation is selected and is induced to fragment further to fragmentation is selected and is induced to fragment further to
give daughter ions
In a complex mixture these daughter ions provide a unequivocal
evidence for the presence of the known compound evidence for the presence of the known compound
Very powerful screening tool
No need for chromatographic purification of complex mixtures
E.g. Urine analysis of humans or race horses to detect presence
of drugs can be done accurately.
In triple-quad,three quadrupoles are used in series (Tandem MS) p q , q p ( )
First quadrupole selects a specific ion of interest,
second induces collisions using RF only, and third separates the
product ions for recording spectrum of daughter ions product ions for recording spectrum of daughter ions.
The three operations can be done simultaneously in an Ion Trap.
Interpretation of
mass spectra
Interpretation of EI spectra
Base
Molecular ion
70eV
Base
peak
[M+1]
In EI spectra the molecular ion peak is usually the peak of highest mass number
(except for the isotope peaks) but M+ could be the weakest peak as well.
The Molecular Ion Peak The Molecular Ion Peak
In EI spectra sometime the fragmentation is In EI spectra, sometime the fragmentation is
so much that the molecular ion peak is missing
How would you ascertain that it is a molecular y m
ion peak? or an impurity? or a fragment?
Simplest way is to record the spectrum again p y p g
in CI mode. You will get an intense peak at
[M+1] with very little and weak fragment
k peaks
Other way to rule out is - apply Nitrogen
R l Rule
The Nitrogen Rule - generalization The Nitrogen Rule generalization
The molecule of even numbered molecular weight
must contain either no nitrogen or an even number of must contain either no nitrogen or an even number of
nitrogen atoms; an odd numbered molecular weight
requires an odd number of nitrogen atoms.
f d h DD f N h h If compound has ODD no. of Nitrogen then M+ has
ODD nominal mass.
If compound has EVEN no of Nitrogen then M+ has If compound has EVEN no. of Nitrogen then M+ has
EVEN nominal mass. (Rare case, usually no N)
Holds good for compounds containing C, H, N, O, S, X,
P, B, Si, As & alkaline earths
This rule results from nitrogen having a valence of
three and an even atomic mass (which results in odd three and an even atomic mass (which results in odd
molecular mass).
Corollary: Fragmentation at a single
bond gives an odd-numbered ion
fragment from an even numbered
l l molecular ion, and an even numbered ion
fragment from an odd numbered
l l h ll h ld molecular ion. For this corollary to hold,
the ion fragment must contain all of the
( f ) f h l l nitrogen (if any) of the molecular ion.
Exception: Inorganic compounds does p g p
not follow this rule e.g. NO (m/z 30),
NO
2
(m/z 46)
2
Some clues
I t it f M d d th t bilit f l i Intensity of M+ depends on the stability of mol. ion
1. Aromatic> conjugated alkenes> cyclic > organic sulfides>
normal alkanes> mercaptans. normal alkanes mercaptans.
2. ketones> amines > esters> ethers> carboxylic acids ~
aldehydes ~ amides ~ halides.
[M-15] peak (loss of CH
3
) or [M-18] peak (loss of H
2
O) Or
[M-31] peak (loss of OCH
3
from methyl ester) is taken as
confirmation of molecular ion peak confirmation of molecular ion peak.
Peaks at M-1 (loss of hydride), M-2 (loss of hydrogen by
thermolysis/fragmentation) or M-3 (from alcohols) are
common.
However peaks between M-3 to M-14 indicate impurity
peaks or a fragment ion Peaks M-19 to M-25 are unlikely peaks or a fragment ion. Peaks M 19 to M 25 are unlikely
except for fluorinated compounds F= 19, HF = 20.
Unlikely masses 4-14, 21-25, 33, 37, 38
Isotope peaks [M+1,M+2.] Isotope peaks [M 1,M 2.]
For example
CO N CH N &C H
HRMS
CO, N
2
, CH
2
N &C
2
H
4
M. Wt. = 28
The sum of formula
masses of the most
abundant isotope gives
HRMS
For CO
1 12 000 1 15 9949 1x12.000 + 1x15.9949 =
27.9959
For N
2
14.0031 x 2 = 28.0062
For CH
2
N
1x12.0 + 2x1.00783 +
1x14.0031 = 28.0187
For C H For C
2
H
4
2x12.0+4x1.00783 =
28.0312.
Atomic weight is however
th f ll th the average of all the
isotopes
Index of Hydrogen Deficiency/
Sit (D ) f U t ti Sites (Degrees) of Unsaturation
It is the no. of pairs of hydrogen atoms that must be It is the no. of pairs of hydrogen atoms that must be
removed from the corresponding saturated formula
to get the compound of interest.
The index is, the sum of the number of rings, the h n s, th sum of th num r of r ngs, th
number of double bonds and twice the number of
triple bonds. (benzene =4, nitro = 1)
Index for C
n
H
m
X
x
N
y
O
z
can be calculated from eqn.
n m x y z
q
Index = (n) (m/2) (x/2) + (y/2) + 1
Calculate for C
7
H
7
NO
7 3 5 + 0 5 + 1 = 5 7 3.5 + 0.5 + 1 = 5
The index give some idea about the structure of
molecular ion. Even-electron ions give an odd multiple
of 0 5 while odd electron fragment ions give integer of 0.5, while odd electron fragment ions give integer
values of the index.
Determination of Formula Determination of Formula
The Rule of 13
A C H d i l t (13 f 1) i Assumes C
n
H
n
and amu equivalent (13 for n=1) is
present in all molecular fragment ions
Step 1: Divide M+ mass by 13 this gives n Step 1: Divide M+ mass by 13, this gives n
Step 2: Any remainder represents count of additional Hs
example 1: for M+ = 78 mp f
78 13 = 6 n = 6 C
6
H
6
example 2: for M+ = 92
92 13 = 7.077 n = 7
7 x 13 = 91 1 extra H is present
F l i C H C7H8 Formula is C
7
H
7+1
= C7H8
example 3: for M+ = 161
161 13 = 12.385 n = 12
12 x 13 = 156 5 extra Hs are present 12 x 13 = 156 5 extra Hs are present
Formula is C
12
H
12+5
= C
12
H
17
Wh t b t h t t ? What about heteroatoms?
Step 1: First derive formula as above
Step 2: Next, modify using C
n
H
m
equivalents p , y g
n m
q
Element C
n
H
m
equiv. Element C
n
H
m
equiv.
1
H12 C
31
P C
2
H
7
16
O CH
4
32
S C
2
H
8
14
N CH
2
16
O
32
S C
4
16
O
14
N C
2
H
6
35
Cl C
2
H
11
19
F CH
7
79
Br C
6
H
7
28
Si C
2
H
4
127
I C
10
H
7
example 4: for M+ = 108
108 13 = 8 308 n = 8 108 13 8.308 n 8
8 x 13 = 104 4 extra Hs are present
Formula is C
8
H
8+4
= C
8
H
12
Possible candidates with heteroatoms
C
8
H
12
CH
4
+ O = C
7
H
8
O
C
8
H
12
2(CH
4
) + O
2
= C
6
H
4
O
2
C
8
H
12
CH
4
CH
2
+ O + N = C
6
H
6
ON (even number?)
Nitrogen rule? Says M+ cant be even numbered.
So, C
8
H
12
2(CH
2
) + N
2
= C
6
H
8
N
2
is possible M+
MS will only give you molecular weight data.
One must use other spectral techniques to get p
information about the presence of heteroatoms.
CHN analysis, IR frequencies, NMR chemical shifts
Problem
The exact mass of a compound determined by
HRMS is 212.0833. What is the molecular
formula of the compound?
H 1 00783 C 12 0 O 15 9949 N 14 0031 H 1.00783, C 12.0, O 15.9949, N 14.0031
212 (12%), 167 (4%), 105 (100%), 91 (45%), 77
(30%) 63 (10%) 51 (12%) St t ? (30%), 63 (10%), 51 (12%). Structure?