Instrumental Methods of Analysis (IMA)

FTIR-SPECTROSCOPY

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 AIM
To analyze the functional groups of the given compound (cinnamic acid
or paracetamol) using IR spectroscopy and identify characteristic peaks
corresponding to their molecular structures.
WORKING PRINCIPLE
 What is Spectroscopy?
Spectroscopy is the study of interaction between matter and electromagnetic radiation
e.g., IR Spectroscopy is concerned with the study of absorption of infrared radiation, which causes vibrational
transition of bonds in molecules. So, it is also called vibrational spectroscopy .

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 What is Spectroscopy?
IR Spectroscopy mainly used to
• Identify the functional groups of the molecule
• Determining the chemical composition of materials.
• Atomic arrangements of a molecule
• Analyzing mixtures to quantify individual components.

Q. Why NIR & FIR are not used in IR Spectroscopy???

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 IR Region
In Spectroscopy:
•Wavelength (λ) is the
Far IR
distance between
Near IR Mid IR successive peaks of a wave.
•Wavenumber (ν) is the
Wave Length: 0.8 – 2.5 μm Wave Length: 2.5 – 50 μm Wave Length: 50 – 1000 μm number of wave cycles per
Wave number: 12,500 – 4,000 cm-1 Wave number: 4,000 – 200 cm-1 Wave number: 200 – 10 cm-1 unit distance.

Interfered with UV FIR is also closer to the radio-wave

radiation and gives extra region and sometimes interact & form
band on IR spectra due extra unwanted band spectra on the IR
to delocalization of spectrum.
electron

MIR is appropriate for the IR spectroscopy. Because it gives fully IR peak on IR spectrum.
So, We used 2.5 – 5.0 μm wavelength in IR Spectroscopy

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Principle of IR Spectroscopy
When IR radiation interact with sample molecule, the sample molecule will absorb the specific
applied quantized IR light. Absorption of IR radiation causes internal vibration of bonds in the
molecule like Stretching and bending vibration. The transmitted light is detected by the
detector and IR spectrum interpreted on the computer screen by analysis of the transmitted
light.
Q. Why is this an inverted graph??

IR Radiation
Sample
(Mid IR)
(absorbs Specific frequency)
Transmitted Light
Range: 4000 – 400 cm-1

Stretching & Bending Vibration

% 𝑜𝑓 𝑇

Wave number, cm-1

 Molecular
Vibration

Stretching Vibration Bending vibration

Higher Frequency Lower Frequency
Changes in bond length Changes in bond angle

Symmetric Asymmetric In-Plane Out of Plane

Stretching Stretching Bending Bending

Scissoring Bending Rocking Bending

Twisting Bending Wagging Bending

NOTE: No. of bending vibration is usually more than the no. of stretching vibration in IR spectrum.
a) Stretching Vibration involves a continuous change in interatomic
distance along the axis of bond between 2 atoms.

In Symmetric Stretching, the bond length increase & decrease

symmetrically.
In Asymmetric Stretching, length of one bond increase & the other
one decrease at the same time without changing bond angle.

b) Bending vibration involves the change of angle between two atoms

Scissoring is the movement of two atoms toward and away from each
other. So bond angle decrease and increase with time.

Rocking in which the bond angle is maintained but bonds moves within
the same plane. Changing angle b/w a group of atoms.

Wagging in which both atoms move to one side of the plane. Changing
angle b/w the plane a group of atoms

Twisting in which one atom is above the plane and the other is below
the plane. Changing angle b/w the plane 2 groups of atoms
 Interpreting an IR Spectrum?
IR Spectrum is the graphical plot of % transmittance verses vibrational frequency in
wavenumbers.
IR Spectrum

%T
Functional Group Region Finger-print Region
4,000 – 1500 cm-1 1500 – 500 cm-1

Identifies the functional group with the Identifies the exact molecule with the
consequence of changing stretching vibration consequence of changing bending vibration
Wave Number, cm-1
Only stretching mode of vibration, so this Stretching and bending both are occurred,
𝐼 𝐼𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦 𝑜𝑓 𝑇𝑟𝑎𝑛𝑠𝑚𝑖𝑡𝑡𝑒𝑑 𝑙𝑖𝑔ℎ𝑡
T= = Intensity of incident light region contains few peaks. but bending is more than stretching
𝐼∙

The pattern of peaks is easy to analyze The pattern of peaks is more complicated
because stretching vary within a narrow due to complex vibration which create a
range. unique IR band to distinguished compounds.

Sharp, Broad, Strong, Medium Peaks Sharp & Weak Peaks

 Peaks
of Functional Group

Shape Intensity
(Width of peak)
(height of peak)

Strong A strong band covers most of the y-axis

Broad Sharp
Medium A medium band falls to about half of the y-axis
Due to H-bonding or
Presence of water Weak A weak band falls to about one third or less of the y-axis
molecules

Factors affecting the Peak intensity :

Electronegativity difference α Polarity α Dipole moment α More Intense Peak

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 How to interpret / analyze IR spectra??
IR spectra isn’t generally used to determine the whole structure of an unknown molecule. But IR
spectra is great for identifying certain specific groups.
Here an overview of the IR window from 4000 to 500 cm-1 with various region of interest highlighted.

Basically 80% of the most useful information for our purpose can be obtained by looking at 3600 to 1600 cm-1 specific
area of spectrum. Remaining peaks from 500 – 1400 cm-1 (fingerprint region) are comparatively less useful.
Like a fingerprint, no two unique molecular structure will produce the same IR spectrum.

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An even more compressed overview looks like this…
NO. Peak take place Common Symbols of Examples of bond of
between (cm-1) bond functional group

ZONE 1 3600 – 2700 X –H O-H bond, C–H bond, N-H bond

ZONE 2 2700 – 1900 X≡X C≡N or C≡C bond

ZONE 3 1900 – 1500 X=O, X=X C=O bond, C=C bond

ZONE 4 1500 -- 1000 X –O , X –X C-O bond, C – C Bond

Here, X symbol will be Oxygen [O] or Carbon [C] or Nitrogen [N]

For Zone 1:
a) O-H bond stretching at 3650 – 3200 cm-1 with strong intensity & broad ‘U’ elongated Shape

b) Terminal alkynes (≡C–H bond) stretching at 3340 – 3250 cm-1 with stronger & sharper peak
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 c) N-H bond stretching mainly occurred for Primary & Secondary amine:
For primary amine, stretching at 3500 – 3200 cm-1 with two signal. These are
medium in intensity & broad shape.
For Secondary amine, stretching at 3500- 3200 cm-1 with one signal . These are weak
intensity & sharp shape

d) =C–H Stretching at 3100 cm-1 with weak intense & sharp peak

e) - C–H Stretching at 2900 – 2800 cm-1 with medium intense & sharp peak

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For Zone 2:

a) C≡C bond stretching at 2200 – 2000 cm-1 with variable intense & shape peak

b) C≡N bond stretching at 2260 – 2220 cm-1 with variable intense & shape peak

For Zone 3:

a) C=O bond stretching peak

for aldehyde at 2200 – 2000 cm-1 , Ketone at 1700- 1725 cm-1, Carboxylic acid at 1715 cm-1, Ester at 1750 – 1735 cm-1
b) C=C bond stretching peak at 1660 – 1600 cm-1

c) C=C bond stretching for aromatic compound peak at 1510 – 1450 cm-1

For Zone 4:
a) C–C bond stretching: peak at 1200 – 1000 cm-1

b) C–O bond stretching: peak at 1150 – 1000 cm-1

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FTIR spec
Fourier transform: mathematical model to breakdown a complex signal (eg. a waveform into
simple sine and cosine waves) – Imagine breaking down music from an orchestra into chords
produced by individual instruments >> into sound waves of different frequencies.

But sample preparation

 ATR-IR spec
• Attenuated total reflectance (ATR) is a
sampling technique used in conjunction
with infrared spectroscopy which
enables samples to be examined
directly in the solid or liquid state
without further preparation
• An attenuated total reflection accessory
operates by measuring the changes
that occur in a totally internally
reflected infrared beam when the beam
comes into contact with a sample.
 Recall the concept of total internal reflection

Q. Can you point out similarities or differences between the two instruments working on the same principle ?
 Materials and Methods
•Cinnamic acid or Paracetamol sample
•Nitrile gloves (for handling samples)
•Tissue paper or kimwipe (for cleaning ATR crystal)
• Isopropyl alcohol (for cleaning ATR crystal)
• Spatula (for loading sample)
•ATR-IR spectrometer
•Opus software for data acquisition and analysis
 Protocol
1.Instrument Preparation
1. Ensure the ATR-IR spectrometer is clean and properly calibrated.
2. Launch the Opus software and set the desired parameters: resolution (4 cm⁻¹), spectral range
(4000–400 cm⁻¹), and scan count (e.g., 25 scans for optimal signal-to-noise ratio).
2.Sample Loading
1. Clean the ATR crystal with IPA and wipe with a kimwipe.
2. Place a small quantity of cinnamic acid/paracetamol on the ATR crystal. Ensure good contact
between the sample and the crystal surface. Clean the crystal again and repeat the process for
second sample.
3.Data Acquisition
1. Record the spectrum for analysed sample. Save the file in Opus Software.
4.Baseline Correction and Analysis
1. Use the Opus software to perform baseline correction and identify characteristic peaks (Peak
calling).
2. Record and compare the obtained spectra with literature data to assign functional groups.
5.Repeatability
1. Repeat the measurements for each sample to ensure reproducibility.
OBSERVATION
Example: Spectrum interpretation for nitroacetophenone
C:\OPUS_7.2.139.1294\MEAS\4-nitroacetophenone.0 9/2/2024 11:43:09 AM
100
99
98
97
96
95

3931.75
3884.46
3850.82
3833.68
3747.34
3703.26
3678.36
3658.73
3637.67
3614.21
3577.46
3534.07
3513.45
3434.35
3224.47
2921.36
2853.54

2311.18

1924.75
1875.47
1835.83
1781.40
1743.24
1684.81
1653.34
1523.04
1461.76
1426.98
1330.59
1232.79
1096.08
94

994.59
948.74
844.69
733.65
682.09
623.36
3500 3000 2500 2000 1500 1000
Wavenumber cm-1

Page 1 of 1

Note the wavenumbers of the characteristic absorption peaks

from the spectra of your given compound.
 RESULTS

Cinnamic acid

Q. Can IR-spec be used to identify a known

compound ?? What about an unknown compound ??

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Application of IR Spectroscopy
i. Identification of an organic compound
ii. Structure determinations
iii. Identification of functional group
iv. Studying the progress of reaction
v. Distinction between two types of hydrogen bonding
vi. Study of keto-enol tautomerism
vii. Study of Cis-trans Isomerization
viii. Conformation analysis
ix. Determination of impurities in compound
x. Presence of water in sample
Limitations of IR Spectroscopy
a. Can’t determine the molecular weight of the compound.
b. Doesn’t give information about the relative position of different functional
groups in a molecule.
c. From the single IR spectra of an unknown substance, it is not possible to know
whether it is pure compound or a mixture of compound.

Q. Is it classified as a qualitative or quantitative technique ??

Q. What precautions can you think of ??

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 If this is too much reading, watch this:
infrared-spectroscopy-characterization-of-functional-groups
Thank You…