CHM 420: Structural Methods and Analysis

Identification/Characterization of Organic Compounds

(small molecules and biomolecules)
IR
UV-visible
Fluorescence
Mass Spectrometry
Elemental analysis (CHN)
NMR
Total: ~41 h
IR Spectroscopy (SGS)
Topics: Principles (Selection Rules and Hooke's Law; Characteristic Group Vibrations),
instrumentation, effect of solvents and substituents, analysis of spectra.

UV-Visible Spectroscopy and Fluorescence Spectroscopy (SGS)

Topics (UV-Visible): Interaction of electromagnetic radiation with molecules,
Instrumentation; Principles (Selection Rules and the Beer-Lambert Law; Chromophores),
different types of transitions
pH, solvent and substituent effects and Applications.
Topics (Fluorescence): Basic principle; factors influencing fluorescence properties.
Solvatochromic fluorophores; conformational sensitive fluorescent molecules; probes for
diagnosis.

Mass Spectrometry (SGS)

Topics: Methods of ionization and separation of ions, detection; main pathways of
fragmentation by electron impact ionization; Different techniques of mass analysis.
Mass spectrometry of biomolecules.
Nuclear Magnetic Resonance (PT)
Topics: Instrumentation; Origin of the NMR Effect; Chemical Shift; Splitting; 1H NMR
Spectroscopy; Decoupled spectra; 1D NOE; 13C NMR Spectroscopy; The principles of two-
dimensional spectroscopy; Correlation 2D spectroscopy (COSY, HMQC, HMBC); Nuclear
Overhouser effect (nature, practical consequences); homo- and hetero nuclear effect;
technique of measurement; difference spectra; sample requirements; Other Nuclei.
Structure Determination (PT)
Topics: Problem solving sessions using 1H, 13C NMR, Mass, CHN, IR data of known and
unknown samples.

Books:
1. Spectrometric Identification of Organic Compounds, R. M. Silverstein, F. X. Webster, D. J.
Kiemle, 7th Ed. 2005, John Wiley & Sons. (IR, NMR and Mass)

2. Spectroscopy by Pavia, Lampman, Kriz, Vyvyan, Cengage Learning, 2007.

3. Organic spectroscopy by William Kemp, 3 rd Ed., Palgrave, 1991 (UV and NMR).

4. Principles of fluorescence spectroscopy by Lakowicz, 2 nd Ed., Springer, 2004 (Fluorescence)

Literature ref. will also be provided

Evaluation procedure
Mid-sem examination: 35 %
End-sem examination: 35 %
Quiz 1/Assignment 1: 10 %
Quiz 2/Assignment 2: 20 %
 How can we characterize molecules?

We can utilize inherent properties of a molecule, which

respond to an external energy source (e.g., when
irradiated with electromagnetic radiation)
 Electromagnetic Spectrum
• The electromagnetic spectrum is divided into different regions, ranging
from gamma rays to radio waves
terahertz

NMR rotational IR UV-Vis crystallography

In terms of size:
What Can Spectroscopy Measure?

molecular Radioactive
structure nuclei
in gas phase (radionuclides)
molecules
 Absorption Spectroscopy
• When electromagnetic radiation falls on a molecule, certain wavelengths
are absorbed and others are virtually unaffected

• For absorption to occur, the energy of photons should match the difference
in energy between the two energy states (ground state and excited state)

• The larger the energy difference between two states, the higher the energy
of radiation needed for absorption

Example
• Electronic excitation: UV-visible light, higher energy
• Vibrational excitation: Infrared, lower energy
 Absorption Spectroscopy (IR and UV-Vis)

Potential energy diagram of diatomic molecule

100-800 nm
Transition takes between
electronic energy levels of non-
bonding and π electrons
Transition is very fast (~10 -15 s) Emission
from
electronic
excited
1000-0.8 μm
state
Transition takes
between vibrational 1 mm-1 m
energy levels
 IR Spectroscopy

1000-0.8 μm 800-200 nm
At temperatures above absolute zero, all the atoms in molecules are in
continuous vibration with respect to each other. When the frequency of
a specific vibration is equal to the frequency of the incident IR radiation,
the molecule absorbs the radiation.
How one could measure this?
 IR Spectroscopy
IR absorption spectroscopy relies on the absorption of specific
frequencies by an organic molecule that are characteristic of bond or a
group of vibrating system.
The absorption frequency depends on the relative masses of the atoms,
force constants of the bonds, geometry of the atoms and the associated
vibronic coupling.
Although the vibrational levels are quantized, vibrational spectra appear
as bands rather than lines because a single vibrational energy change is
accompanied by several rotational energy changes. So it is a combination
of vibrational-rotational bands we observe in IR spectra.
 IR is divided into 3 regions

hc
E = hν = => ν= c c = ~3 x 1010 cm/s
λ λ

Increase in wave number corresponds to an increase in energy

Harmonic oscillator Based on Hooke’s law
composed of 2 masses
(here atoms) Quantum mechanical model: Vibrational
Classical model motions are quantized (divided into energy
levels).
rotational transitions
(microwave region)

ν=3

Potential Energy (E)

ν=2

ν = 1 E1 = 3/2hν
Vibrational transitions
ν = 0 E0 = 1/2hν

Inter-atomic Distance (r)

E=½ kx2
E = (n + 1/2)hν
where: x is spring displacement
where: ν is the frequency of the vibration,
k is spring constant
n is the quantum number (0, 1, 2, 3, . . . )
m is reduced mass
The energy or frequency is dependent on how far one stretches or
compresses the spring, which can be any value. Thus a molecule
could absorb energy of different wavelengths.
 Anharmonic oscillator
Bonds can break and bonds can be compressed to a certain point only.
Hence, molecules are anharmonic oscillators

• The lowest energy level is E0 = 1/2 hν.

• The next highest is E1 = 3/2 hν.
• Selection rule: Transitions to the next
energy level are allowed: transitions
of energy hν are mostly observed
E1 = 3/2hν
E0 = 1/2hν
• Occasionally transitions of 2 hν, 3 hν, or
higher are observed.
• These correspond to bands called
overtones in an IR spectrum. They are of
lower intensity than the fundamental
vibration bands.
hc
E = hν = => ν= c c = ~3 x 1010 cm/s
λ λ Increase in wave
number corresponds to
an increase in energy
Degree of Freedom
Degree of freedom is the number of variables required to describe the motion of a
particle completely. For an atom moving in 3-dimensional space, three coordinates
are adequate, so its degree of freedom is three. Its motion is purely translational. If
we have a molecule made of N atoms (or ions), the degree of freedom becomes 3N,
because each atom has 3 degrees of freedom. Furthermore, since these atoms are
bonded together, all motions are not translational; some become rotational, some
others vibrational. 3 degrees are required to describe translation motion. For non-
linear molecules, all rotational motions can be described in terms of rotations around
3 axes, the rotational degree of freedom is 3 and the remaining 3N-6 degrees of
freedom constitute vibrational motion. For a linear molecule however, rotation
around its own axis is no rotation because it leave the molecule unchanged. So there
are only 2 rotational degrees of freedom for any linear molecule leaving 3N-5 degrees
of freedom for vibration.
Fundamental Modes of Vibrations
Consider a molecule containing “n” atoms:
Position of each atom relative to other atoms can be specified by three
coordinates (x, y and z Cartesian coordinates)
For n atoms: 3n coordinate values (the molecules has 3n degrees of freedom
including translation, rotation and
vibration)
For a non-linear molecule:
3 degrees are required to describe translation motion and 3 degrees for
rotational motion
Remaining 3n-6 are Vibrational degrees of freedom
For a linear molecule:
3 degrees are required to describe translation and 2 degrees for rotational
motion (rotation about the molecular axis does not involve movement of any atomic nucleus)
Remaining 3n-5 are vibrational
Number ofdegrees of freedom
Fundamental Vibrations
Two types of molecular vibrations:
Stretching and Bending

Not all stretching and bending vibrations are observed in

the IR spectrum

Fundamental vibrations that produce a rhythmic change in

the dipole moment are IR active

Alternating electric field produced by the changing charge distribution

accompanying the vibration couples with the electric field of the
incident radiation
 Stretch – Vibration or oscillation along the line of the bond

H H
C C
H H

symmetric asymmetric

Bend – Vibration or oscillation not along the line of the bond

H H H H
C C C C
H H H
H
scissor rock twis wag
in plane t out of plane
Scissoring Rocking Twistin Wagging
g
Two types of molecular vibrations: Stretching and Bending
Not all stretching and bending vibrations are observed in the IR spectrum

e.g., long chain hydrocarbons

 CO2 is linear and hence has 4 fundamental vibrations (3n-5)

Symmetric stretching:
no change in the dipole
moment: IR inactive

Bending vibration: IR active

Asymmetric stretching: IR active
CO2 is linear and hence has 4 fundamental vibrations
Observed two bands

(seen in Raman)

Center of mass changes

In these cases

H2O/CO2 = 5 ice mixture at 15 K.

Example: HO2 : nonlinear, has three fundamental vibrations
(3n-6)

Energy required to stretch a

bond is more than to bend the
bond

Wave number will be higher

for stretching vibrations than
bending vibrations
One can calculate the vibration frequency (stretching) with reasonable
accuracy by using Hooke’s law
1 amu (dalton) = 1 g mol-1
= 1.6605 x 10-24 g

Example: C-H bond stretching

f = 5 x 105 dyne/cm
MC = 20 x 10-24 g
MH = 1.66 x 10-24 g

Calculated: 3032 cm-1

Observed: 3000-2850 cm-1
Absorption frequency depends on the bond strength and mass of the atoms
e.g., mass of atom is increased
Bond type Force constant Calculated Observed
Dyne/cm cm-1 cm-1
Bond strength ν

5.0 x 105 3032 3000-2850

Mass of atom ν
5.0 x 105 2225 2250-2080

4.5 x 105 1128 1300-800 1 dyne/centimeter = 0.001 Newton/meter

 Stronger the bond, higher is the force constant and higher the
wavenumber
Example: for atoms of same mass
C≡C bond is stronger than C=C bond is stronger than C-C bond
Bond type Force constant Calculated Observed
Dyne/cm cm-1 cm-1

15.6 x 105 2101 2150-2100

Same mass but 9.7 x 105 1657 1900-1500
Diff. force
const. 4.5 x 105 1128 1300-800

4.9 x 105 1135 1250-1000

Similar mass and
5.0 x 105 1113 1300-800
similar force const.
7.0 x 105 3553 3800-2700

Here mass difference 12.1 x 105 1731 1850-1600

plays a major role
When an atom of higher mass is present;
the trend is similar (force constants are different)
Bond length
(0.214 nm) C─I 500 cm-1
(0.193 nm) C─Br 600 cm-1
(0.177 nm) C─Cl 750 cm-1

(0.143 nm) C─O 1110 cm-1

(0.154 nm) C─C 1130 cm-1

(0.109 nm) C─H 3030 cm-1

A smaller atom therefore gives rise to a

higher wavenumber and a larger atom
gives rise to a lower wavenumber

Based on bond length

O-H stretching ?
 Group frequencies

vibration frequency range cm-1) vibration frequency range (cm-1)

carbonyl (C=O) stretch 1870 - 1650 Amines
Alcohols N-H stretch 3460 - 3280
O-H stretch 3640 - 3250 NH2 wag 1650 - 1590
C-OH stretch 1160 - 1030 C-N stretch 1190 - 1130
C-OH in-plane bend 1440 - 1260 C-N-C scissor 510 - 480
C-OH wag 700 - 600 Esters
Alkanes C-O-C asymmetrical stretch 1290 - 1180
C-H stretch 2980 - 2850 O-C-O scissors 645 - 575
CH2 wag 1470 - 1450 Nitro compounds
CH2 rock 740 - 720 NO2 symmetrical stretch 1570 - 1550
CH3 wag 1390 - 1370 NO2 asymmetrical stretch 1380 - 1360
CH3 twist 1470 - 1440 NO2 scissors 650 - 600
Alkenes NO2 rock 530 - 470
=CH2 stretch 3040 - 3010 Sulfones
=CH2 wag 950 - 900 SO2 symmetrical stretch 1170 - 1120
C=C stretch (cis) 1665 - 1635 SO2 asymmetrical stretch 1360 - 1290
C=C stretch (trans) 1675 - 1665 SO2 scissors 610 - 545
 The intensity of an IR band is affected by two factors
• Whether the vibration is stretching or bending
Dipole moment will change more for stretching than bending: Stretching will
be usually more intense
• Difference in the electronegativity of the atoms involved in the bond
Greater the difference in electronegativity between the atoms involved in
bonding, the larger the dipole moment change: so more intense the peak

It is important to make note of peak intensities to show the effect of these

factors:

Types:
Strong (s) – peak is tall, transmittance is low
Medium (m)
Weak (w) – peak is short, transmittance is high
Broad (br)
Example of an IR spectrum to show the types of bands

br
s
s
 Factors Influencing the Vibration Frequencies
In a polyatomic molecule, no vibration occurs without affecting or getting
affected by adjoining vibrating bonds.
This results in mixing and redistribution of energy states, yielding new
energy levels and different vibrational frequencies.

Example
Difficult to predict
Based on the mass

Factors that can alter the position, intensity and appearance of IR bands
1. Vibrational coupling
mechanical coupling
Fermi resonance
2. Hydrogen Bonding
3. Electronic Effects
4. Ring Strain
5. Solvent
Vibrational coupling
When two oscillating bonds share a common atom then there can be
mechanical coupling and the bonds seldom behave as individual oscillator.
Coupling of two fundamental modes will produce two new vibrations
(symmetric and asymmetric) with frequencies higher and lower than if
coupling is absent.
Interaction is higher if the coupled groups absorb individually near the same
frequency (e.g., C-C and C-N are similar 1130 cm-1).

coupled oscillators:
https://www.youtube.com/watch?v=CguKKl9mX2s
2980–2850 cm-1 https://www.youtube.com/watch?v=izy4a5erom8
Vibrational coupling
Interaction is higher if the coupled groups absorb individually near the same
frequency (e.g., C-C and C-N are similar 1130 cm-1).
 Vibrations of similar frequencies couple better and often show higher stretching frequency

~1715 cm-1
Ineffective coupling
IR inactive Lower than CO2
IR active

Higher absorbing band 1800–1900 cm-1

but this is less than C=O in CO2?
C=O stret
Primary and secondary amines

3500–3080 cm-1
Coupling interactions:
Coupling between stretching and bending vibrations can occur if the stretching bond is
part of one of the bending bond atoms.
Eg., amides show two bands ~1500-1650 cm-1
corresponding to C=O stretching and N-H bending
(coupled) and these are designated as amide I and
amide II bands
Here, N-H bending frequency (amide II) is altered by
coupling of N-H bending and C-N stretching vibrations
C-N stret = 1190 cm-1

C=O
N-H stret
stret
N-H bend, amide II
 Fermi resonance
Interactions can occur between a fundamental band and an overtone.

The out of plane bending of aromatic C-H bonds occurs at 865 cm-1; the overtone
(1730 cm-1) of this band coincides with the fundamental of C=O at 1730 cm -1

This Fermi resonance results in two bands (1760 and 1720 cm -1)

Splitting of C=O overtone C-H out of

by Fermi plane bending
resonance
C=O splitting due Fermi resonance can be seen in some cyclic ketones
Requirements for effective vibrational or mechanical coupling

Coupling interaction also depends on the geometry

of the vibrating system
Hydrogen Bonding
One of the most common effects seen in organic compounds:
can change the peak shape and position of IR bands

Formation of H-bond lengthens the original O-H and O-R

bonds, and hence weaken these bonds

This lowers the stretching frequency of both the bonds

Free O-H bond is sharp and comes at higher wavenumber and is usually seen in
vapour phase and dilute solutions (3700-3580 cm-1)

Due to H-bonding, dynamic polymeric structure can be formed which

will widen the envelope of absorption band (bond widening will take
place). Examples alcohols, phenols, carboxylic acids.
H-bonded O-H group is broad and comes at lower wavenumber,
3550-3200 cm-1

Vibration frequency will depend on inter and intramolecular H-bond

Same is true for amines and amides also.
In amines the shift due H-bond is not as dramatic: N is less
electronegative than O
Examples: Intermolecular H-bond
Polymeric structure in alcohols/phenols/acids
Intermolecular H-bond depends on concentration
Alcohols, phenols, acids exist in dynamic polymeric structures due H-bonds

0.03M 1.0 M
3620 cm-1 3330 cm-1
in CCl4
 Intramolecular H-bond is concentration independent

0.03M 1.0 M

3600 cm-1 (sharp for free hydroxyl)

3100 cm-1 (broad for intermolecular H-
bond)
Intramolecular H-bond is concentration independent

In enols O-H str wavenumber

can be as low as 2800 cm -1

1724 cm-1 Not only O-H vibration

1680 cm-1 is affected

Even carbonyl vibration

is affected by H-bond
 Conjugation/Inductive/Resonance Effects
Conjugation reduces the double bond character of C=O and C=C bonds, and hence
the force constant of the bonds: Conjugation reduces C=O and C=C str frequency

Resonance/Mesomeric (R/M) and inductive (I) effect play a major role

Depending on the nature of the group attached to C=O bond, stretching frequency will
be higher or lower as compared to parent carbonyl-containing compound.

In general +R/M effect weakens the bond and lowers the frequency
-I effect strengthens the bond and increases the frequency Inductive effect
(20-30 cm-1 shift)

In a given system it is difficult to isolate the I effect from R effect.

resonance effect
 Conjugation/Inductive/Resonance Effects
Eg., for resonance effect:

Here there is no vibrational coupling

Bond strength of C=O is affected due to resonance

+M EFFECT ORDERS :
–O- > –NH2 > –NHR > –OH >–OR > –NHCOR > –OCOR > –Ph > –F > –Cl > –Br > –I
-M EFFECT ORDER :
–NO2 > –CN > --S(=O)2−OH > –CHO > –C=O > –COOCOR > –COOR > –COOH > –CONH2 > –COO−
Decreasing order of –I effect
—NR3+ > —NO2 > —SO2R > —CN > —COOH > —F > —Cl > —Br > —I > —OR > —COR > —OH > —C6H5 > —CH2=CH2 > —H
Resonance versus inductive effect:

Acyl halides: In this case –I effect of chlorine dominates

Increases C=O str. frequency

Amides: The +R effect of NH2 dominates the –I effect of

nitrogen
weakens the C=O bond and lowers the frequency

Alkyl esters: 1750-1735 cm-1

In alkyl esters, the nonbonding electrons on oxygen
participates in conjugation.
But C=O stret. is higher than ketones, why?

In phenyl esters, nonbonding electrons on oxygen are

partly drawn into the ring, thereby the conjugation effect
is diminished. –I effect of oxygen also contributes, which
increases the frequency of C=O str. as compared to alkyl
esters
 Ring Strain: Ring strain increases the C=O and C=C stretching frequency
As ring strain increases stret. frequency increases
s character of C=O bond increases (i.e. bond strength increases)

1850 1783 1750 1715 1709 cm -1

As O=C-C bond angle increases s-character of the bond increases and hence, strength
increases
sp3 => 109.8o, sp2 => 120o, sp = 180o
As this angle reduces, the coupling with adjacent C-C bond increases
120o and energy required to stretch C=O increases

Same is true for C=C-C in exocyclic alkenes

νC=C 1780 1678 1657 1651 cm-1

Endocyclic: Ring strain shifts the C=C band to lower wavenumber
(except: cyclopropene)

The C=C stretching vibration is coupled with C-C stretching of adjacent bond

νC=C 1650 1646 1611 1566 1641

Solvent effect (range is 25 cm -1):

Fundamental vibrations that can involve in H-bonding can be affected by solvents

In nonpolar solvents the frequency is increased compared to neat sample

In polar solvent due to H-bonding with solvent, the frequency decreases

Steric effects that reduce the coplanarity of the conjugated system reduce
the effect of conjugation (note: conjugation reduces the C=O and C=C str.
frequency)

e.g., α,β-unstaurated ketones: steric hindrance in cis reduces the resonance

in the cis-isomer

H
Both the isomers
exists at RT
H

C=C
more pi conjugated less pi conjugated
Examples

C=O stret
1716 1685 1716 1683 1678
cm-1

1748 1710 1750 1712

1800 1750
 General – Along with alcohols, the most ubiquitous group on the IR spectrum.
Although it is easy to determine if the C=O is present, deducing the exact
functionality and factors that influence the position of the band provide
the challenge

Base C=O Frequencies (cm-1), Stretch (sym.)

C=O 1810 Anhydride band 1

1800 Acid Chloride
1735 Ester
1725 Aldehyde
1715 Ketone
1710 Carboxylic Acid (H-bonding effect is also present)
1690 Amide (H-bonding effect is also present)

For your information:

Decreasing order of –I effect
—NR3 > —NO2 > —SO2R > —CN > —COOH > —F > —Cl > —Br > —I > —OR > —COR > —OH > —C6H5 > —CH2=CH2 > —H