Infrared spectroscopy of

Complex molecules

Dipak K. Palit
 Infrared Spectroscopy of Complex Molecules
➢ Because of 3N-6 or 3N-5 rules, a complex molecule is likely to have an IR spectrum
exhibiting a large number of normal modes of vibrations.
➢ Each normal mode involves some displacement of all or nearly all, the atoms in the
molecule.
➢ However, while in some of the modes, all atoms may undergo approximately the same
displacement, in other modes, displacement of a small groups of atoms (e.g. in a functional
group) may be much more vigorous than those of the remainders.
➢ Thus we divide the normal modes into two classes:
SKELETAL Vibrations: Arise from strong coupling between stretching or bending motions of atoms
in straight chains, branched chains or in a ring. Many atoms involved, all undergoing approximately
the same displacement.
For organic molecules, these vibrations usually fall in the range 600 – 1400 cm-1 and arise
from linear or branched chain structures or rings.

Characteristic GROUP Vibrations: Vibrations that are associated with certain functional groups. It
is possible to identify a functional group of a molecule by comparing its vibrational frequency on an
IR spectrum to an IR stored data bank.
Group frequencies are usually almost independent of the structure of the molecule as a whole
and with a few exceptions, fall in the regions well above and well below that of the skeletal modes .
 n-Hexane
Oop bend of Methyl and
Methylene group: 724, 758 cm-1
In-plane bend of
methyl group: 1378 cm-1
In-plane bend of
methylene group: 1460 cm-1

CH2: Symmetric Stretch: 2850 cm-1; Asymmetric stretch: 2923 cm-1.

CH3: Symmetric Stretch: 2874 cm-1; Asymmetric stretch: 2958 cm-1.

 Symmetric
in-plane Oop CH bends
CH bend

Asymmetric in plane
Coupling in CH2 group C-H bend
1470 cm-1
Ring modes
C=O overtone
 O=C-H bending: 1400 cm-1
Fermi-doublet
Aldehyde C- H stretch
and overtone of C-H
bend.
C = O stretching:1750 cm-1

2700
2800

Overtone and Fermi doublet bands

Normally C-H stretching mode of aldehydes appears in the 2850 – 2720 cm-1 region as Fermi
doublet of medium intensities. This is attributed to Fermi resonance between the fundamental
aldehyde C-H stretch and the first overtone of the aldehyde C-H bending vibration which appears
near around 1400 cm-1. Only one aldehyde stretching band is observed for aldehydes whose C-H
bending band is shifted appreciably from 1400 cm-1.
An absorption of medium intensity near 2720 cm-1 accompanied by a carbonyl absorption band is
good evidence for the presence of an aldehyde group.
Fermi doublet

O=C – H bend
(1410 cm-1)
 a
Aromatic C=H stretch
3030cm-1
Aldehyde
C-H bending

aAldehyde C-H stretch as a Fermi doublet at 2825 cm-1 and 2720 cm-1. In this purely aromatic
aldehyde, the band at 2825 cm-1 is clearly seen, since no overlap with other aliphatic C- H
stretching. The doublet is due to Fermi Resonance between the overtone of the C-H bending
having frequency near 1400 cm-1 and the aldehyde C-H mode near 2720 cm-1.
C = O stretch: 1795 cm-1, Fermi-resonance band at 1745 cm-1 between the C=O stretch and
overtone of 875 cm-1 band (out of plane C-H bend)
Carboxylic anhydrides CH3 bend

Antisymmetric and
symmetric stretches

Aromatic C-H stretch

Coupled C=O stretch

C-O stretch
 C-O stretch

Coupled C=O stretch

Coupled vibrations: NO2 group

1575 and 1380

1523 and 1347

Aliphatic Amines

In-plane

CH3CH2CH2-NH(CH3)
Overtone of N-H bend

3000 1000
 1-butanol

3200-3640 (b) O-H

C-O 1o

CH3CH2CH2CH2-OH
 2-butanol

O-H
C-O 2o
 tert-butyl alcohol

O-H
C-O 3o
Carboxylic Acid
A carboxylic acid functional group combines the features of alcohols and ketones, because it has
both the O-H bond and the C =O bond. Therefore, carboxylic acids show a very strong and broad band
covering a wide range between 2800 and 3500 cm-1 for the O-H stretch. At the same time, they also
show the stake-shaped band in the middle of the spectrum around 1710 cm-1 corresponding to the
C=O stretch.
Coupled vibration
of CO2-
C-H deformation
Esters
 IR spectra of Amides Brian C. Smith: Spectroscopy, Vol.35

Amides are organized into three types: primary, secondary, and tertiary. The difference between
them is the number of C-N bonds, with the three amide types having 1, 2, or 3 of these bonds,
respectively. Correspondingly, the number of N-H bonds goes down as the number of C-N bonds
goes up, with primary amides having an NH2 group, secondary amides having an N-H group, and
tertiary amides having no N-H bonds.

(i) Similar to carbonyl groups attached to a benzene ring, amides undergo conjugation. As a result,
their carbonyl stretch is lower than that of many other similar functional groups, and this peak falls
for all amides from 1680 to 1630. As conjugation takes place in all amides, so they all have the same
carbonyl stretching peak range.

(i) Amides also engage in hydrogen bonding. However, like amines, the strength of the hydrogen
bonding is less than that for O-H bonds, giving medium width and intensity of N-H stretching peaks
compared to O-H stretches.
Important features of the IR spectra of amides:
(a) N – H stretch: (3140 – 3500 cm-1) to the left of the C-H stretch. (Amide A Band)
Primary amides show a dual band, secondary amide single band and tertiary amide no N-H stretch
band.
Two bands in primary amides appear at 3500- 3400 cm-1 in nonpoplar solvents. Secondary
amides(trans-conformation): 3430 cm-1.

Amide B band (about 3100 cm-1) originates from a Fermi resonance between the first overtone of
amide II (N-H bend) and the N-H stretching vibration.
(b) Amide I band: Simple amides show lower C=O stretch frequencies at 1650 – 1690 cm-1. due to
resonance in the amide functional group.
In hydrogen-bonding solvents, C=O stretch appears at 1650 cm-1 and in nonhydrogen bonding
solvents at 1690 cm-1. In tertiary amide, C=O stretch frequency has no effect in hydrogen-bonding
solvents.
(c) Amide II band: N-H bend. Tertiary amide does not show this band. Primary amides: 1640 – 1600 cm1
and secondary amides: 1570 -1515 cm-1.
(d) C- N Stretching: Amide III band. ~1400 cm-1.
 Primary Amides

NH-waging

Amide I Band: C=O stretch In the case of primary amides, the amide II band appears just
Amide II Band: N – H bend to right of the amide I band. For secondary amides, amide II
Amide III Band: C - N stretch band appears at lower frequency than that of primary amides.
 Primary Amides

Primary amides have a structural similarity to primary amines. Primary amines have scissoring
and wagging bending vibrations. The primary amide scissoring peak is seen at 1622, and, in
general, this peak falls from 1650 to 1620. The NH2 wag is a broadened envelope, due to
hydrogen bonding, around 700. This envelope normally tops out between 750 and 600. The pair
of sharp peaks on top of this envelope are the C-H wag and ring bend of a mono-substituted
benzene ring, which is present in benzamide.
Secondary Amide
 Secondary Amide

The C=O and N-H in plane bend peaks form a diagnostic pair of sharp, intense peaks in the
middle of the spectrum. Combine these with the single N-H stretch of secondary amides and you
have a trio of peaks that clearly indicate when a secondary amide is present in a sample.
For the nylon family of polymers particularly, this pair peaks falls at ~1640 and ~1540, and are
easily spotted. The N-H wag of secondary amides forms a broadened envelope from 750 to 680
and is seen at 691.
Tertiary amides.
This amide group has no N-H bonds, hence no N-H stretching and bending peaks,
and hence no useful group wavenumbers. Tertiary amides will exhibit a C=O stretch
from 1680 to 1630 like all other amides, but there are many conjugated C=O
functional groups whose carbonyl peaks also fall in this range, meaning there is
nothing unique about the spectra of tertiary amides. It is an example of a functional
group that are not readily detected by infrared spectroscopy.
Imide

Like that amides have a carbonyl group attached to a nitrogen, an imide, as in the
cases of steroids, it has two carbonyl groups connected by a central nitrogen atom.

The spectra of amides and imides have similarities. Imides also have some similarity
to acid anhydrides. Recall that an acid anhydride is two carbonyl groups connected by
a central oxygen atom. The double carbonyl group an acid anhydrides gives rise to a
double carbonyl stretch. Some imides exhibit this type of spectral feature as well.

Note that the central nitrogen atom in the imide functional group is called the imide
nitrogen. This nitrogen atom may have a hydrogen atom bonded to it, as shown in
Figure 1, or have a carbon bearing substituent such as an alkyl chain or phenyl group
attached. In either case, the molecule is still classified as an imide.
Infrared Spectroscopy of Imides
Phthalimide has a hydrogen attached to the imide nitrogen. Imides like this will exhibit an
imide N-H stretch, which falls at 3200±50 cm-1. The N-H stretching peak position is the same
for both cyclic and straight chain imides.

One of the spectral signatures of a cyclic imide is a double carbonyl stretch at 1774 and 1745. In
general, these two peaks are found from 1790 to 1735, and 1750 to 1680 for cyclic imides. Acid
anhydrides also have a double carbonyl stretch that falls in this range but acid anhydrides do not
contain nitrogen, and hence will not exhibit an N-H stretch. Additionally, acid anhydrides have a
strong C-O stretching peak that is missing from imides, because they do not contain this kind of
bond. Thus, the diagnostic pattern for a cyclic imide is a single N-H stretch (when present) in
combination with a double carbonyl stretch.
Amino Acids

N+-H bend

Amino acids exist as zwitterions, so they can be considered both carboxylate salts and amine
salts.
COO- stretch: Frequency is lowered compared to parent acid, since resonance gives the
carbonyl much more single-bond character. Strong asymmetric at 1600 and strong symmetric
at 1400.
N + -H stretch: Broad stretch at 3300-2600. Ammonium ions absorb to the left of this range,
while tertiary amine salts absorb to the right. A broad peak often appears near 2100.
N + -H bend: Occurs at 1610 to 1500. Tertiary amines absorb only weakly.
N – H stretch

N-H bend
C=O Stretch
Proteins
Proteins are polymers that consist of amino acid repeat units. The repeat units are
linked together by secondary amide linkages.

Protein secondary structure describes the repetitive conformations of proteins and

peptides. There are two major forms of secondary structure, the α-helix and β-sheet,
so named by the patterns of hydrogen bonds between amine hydrogen and
carbonyl oxygen atoms that create the peptide backbone of a protein.
Understanding protein secondary structure is important to gain insight into protein
conformation and stability.

A typical protein infrared (IR) spectrum often contains nine amide bands, with
vibrational contributions from both protein backbone and amino acid side chains.
Among which, of particular pertinence to protein secondary structure are Amide I
and Amide II bands. Since both C=O and N—H bonds are involved in the hydrogen
bonding between different moieties of secondary structure, the positions of both
Amide I and Amide II bands are sensitive to the secondary structure composition of
a protein, although the Amide II band is widely viewed as a less useful predictor for
quantifying the secondary structure of proteins.
The structure of a protein, keratin, is shown here.
Keratin : secondary amide with hydrogen bond linkages

Mammal hair, skin, feathers, and reptile scales are all made from keratin, so it is a commonly
found material. This spectrum is of sheep hair (wool).
The spectrum is dominated by secondary amide peaks, with N-H stretch, C=O stretch, and N-H in-
plane bend peaks clearly visible.
The shifts in the Amide I band are often small compared to the intrinsic width of the band,
resulting in one broad peak instead of a series of resolved peaks for each type of the
secondary structure. Mathematical procedures such as Fourier self-deconvolution and
second derivatives can be used to resolve the overlapping bands for the quantitative
analysis of protein secondary structure. Table 1 shows the secondary structure band
assignments for proteins in water. Note that all assignments are depicted as a range, as the
exact position of each peak varies from protein to protein due to the differences in
hydrogen bonding interactions and the environment of the proteins.
Figure 1: FTIR spectra for cytochrome C in phosphate buffer (cytc_12) at 12
mg/mL and 6 mg/mL (cytc_6), and phosphate buffer blank.
FTIR spectra after the buffer spectrum was
subtracted using PROTA-3S software.
Only those buffers with minimum or no peaks in the Amide I and II region should be
selected. Figure shows the ATR-FTIR spectra of BSA in phosphate buffer, dried on the
crystal from a 1 mg/mL solution. In addition to the Amide I and II bands, there are
spectral features of the side chain, such as 1515 cm-1 from tyrosine and 1498 cm-1 from
aspartic acid. Side chain peaks are critical in the elucidation of protonation and
deprotonation states of various amino acids
Peak deconvolution of the Amide I peak (Figure 4) of BSA was carried out using the OMNIC
software. The deconvolution of Amide I resulted in 5 peaks, and the area under each peak
was then evaluated against the total area. Amide I peak deconvolution shows a secondary
structure composition of 47% α-helix, 3% β-sheet, 24% coils, and 26% random,
 H-bond dynamics in the Hydrogen-bonded complex
Fluorenone forms strong H-bonded complex with alcohols in its
ground electronic state.
v=1 Cyclohexane (1725 cm-1)
1.0 Free: 1721 cm-1
Cyclohexane 1-octanol
0.8
absorbance

1:1 comp: 1713 cm-1

0.6 (a)
1-Octanol
(b) ↓ 1:2 comp: 1700 cm-1
0.4 ↓
(c)
0.2 ↓
v=0
0.0
Vibrational energy lebels of
1680 1690 1700 1710 1720 1730 1740 flurenone in cyclohexane and
-1
wavenumber (cm ) octanol

FTIR spectrum of Fluorenone

 CO stretching vibration

FL FL:MeOH FL:(MeOH)2

1784 cm-1 1761 cm-1 1736 cm-1

Quantum chemical calculation by DFT [B3LYP 6-

31++G(d)]