Infrared Absorption Spectroscopy for B.Sc. Biotechnology

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Infrared Absorption Spectroscopy
1. Introduction
• Infrared (IR) spectroscopy is an analytical technique used to identify and study
chemical substances based on how they absorb infrared light.
• Molecules absorb IR radiation at frequencies corresponding to the vibrations of their
chemical bonds.
• Mainly used for qualitative analysis (functional group identification) and sometimes for
quantitative analysis.
• Infrared (IR) spectroscopy or vibrational spectroscopy is an analytical
technique that takes advantage of the vibrational transitions of a molecule.
• It is one of the most common and widely used spectroscopic techniques
employed mainly by inorganic and organic chemists due to its usefulness in
determining the structures of compounds and identifying them.
• The method or technique of infrared spectroscopy is conducted with an
instrument called an infrared spectrometer (or spectrophotometer) to produce
an infrared spectrum.

2. Principle
• IR radiation causes vibrational excitation of covalent bonds in a molecule.
• A molecule can absorb IR radiation if:
o The vibration results in a change in dipole moment of the molecule.
• Each bond or functional group has a characteristic absorption frequency.
• The relation between frequency (ν), wavelength (λ), and wavenumber (νˉ\bar{\nu}) is:
νˉ=1λ(in cm−1)\bar{\nu} = \frac{1}{\lambda} \quad \text{(in cm}^{-1}\text{)}

3. IR Regions

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Region Wavelength (µm) Wavenumber (cm⁻¹) Application
Near IR 0.78 – 2.5 12,800 – 4,000 Overtone & combination bands
Mid IR 2.5 – 50 4,000 – 200 Fundamental vibrations (most common)
Far IR 50 – 1,000 200 – 10 Heavy atom vibrations & lattice vibrations


4. Molecular Vibrations
Two main types:
A. Stretching Vibrations
• Change in bond length.
• Symmetric Stretching: bonds lengthen/shorten together.
• Asymmetric Stretching: one bond shortens, other lengthens.
B. Bending Vibrations
• Change in bond angle.
• Types:
o Scissoring (in-plane)
o Rocking (in-plane)
o Wagging (out-of-plane)
o Twisting (out-of-plane)

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5. Instrumentation

Source → Sample Cell → Monochromator → Detector → Amplifier → Recorder
1. Radiation Sources:
• Nernst glower (ZrO₂ + Y₂O₃ + ThO₂)
• Globar (silicon carbide)
• Incandescent wire (nichrome)
2. Sample Cells & Holders:
• NaCl, KBr windows (transparent to IR)
• Gases in long path cells
• Liquids between IR-transparent plates
• Solids as KBr pellets or thin films
3. Monochromator:
• Grating or prism to disperse IR light.

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4. Detector Types:
• Thermocouples
• Thermistors
• Golay cells
• Pyroelectric detectors
5. Readout System:
• Converts detector signal to an IR spectrum (absorbance vs. wavenumber).

6. Sample Preparation
• Gases: Confined in gas cells with IR-transparent windows.
• Liquids: Sandwiched between NaCl plates.
• Solids: Ground with KBr powder → pressed into a transparent pellet.

7. Interpretation of IR Spectrum
• X-axis: Wavenumber (cm⁻¹) decreasing from left to right.
• Y-axis: % Transmittance (or Absorbance).
• Functional group region: 4000 – 1500 cm⁻¹
• Fingerprint region: 1500 – 500 cm⁻¹ (unique for each compound)
Characteristic IR Absorption Frequencies:
Functional Group Wavenumber (cm⁻¹)
O–H stretch (alcohol) 3200 – 3550
N–H stretch (amine) 3300 – 3500
C=O stretch (carbonyl) 1650 – 1750
C–H stretch (alkane) 2850 – 2960
C≡C stretch (alkyne) 2100 – 2260
C≡N stretch (nitrile) 2220 – 2260

8. Applications
• Identification of functional groups in organic compounds.
• Structural elucidation of unknown molecules.

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• Detection of impurities.
• Monitoring chemical reactions.
• Polymer analysis.
• Quality control in pharmaceuticals & chemicals.

9. Advantages
• Non-destructive technique.
• Requires only a small sample.
• Provides specific information about functional groups.
10. Limitations
• Cannot detect symmetrical diatomic molecules (e.g., N₂, O₂).
• Complex mixtures may give overlapping peaks.
• Requires IR-transparent materials for sample holders.
Diagram Example – IR Spectroscopy Setup