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Infrared (IR) Spectroscopy: Overview

Infrared (IR) Spectroscopy: Overview

When electromagnetic radiation passes through a material, atoms or molecules transition from a lower to a higher energy state by absorbing radiation corresponding to the energy difference between the two states. The absorption of infrared (IR) radiation causes transitions between vibrational energy levels in a molecule. Therefore, IR spectroscopy is a useful analytical tool for determining the molecular structure of molecules.
Different compounds display unique properties due to their...

Applications of IR Spectroscopy: Overview

Applications of IR Spectroscopy: Overview

The non-destructive nature and ability to provide valuable chemical information make IR spectroscopy a versatile technique with broad applications in various scientific and industrial fields. IR spectroscopy is commonly used to identify and characterize organic and inorganic compounds. It provides information about the functional groups present in a molecule and the bonding between atoms. This helps in the structural elucidation of compounds during organic synthesis, pharmaceutical research,...

IR Frequency Region: Fingerprint Region

IR Frequency Region: Fingerprint Region

IR spectra are divided into two main regions: the diagnostic region and the fingerprint region. The diagnostic region of the spectrum lies above 1500 cm−1. The absorptions resulting from single-bond vibrations of the N–H, C–H, and O–H stretch at higher wavenumbers and appear on the left side of the spectrum. The stretching absorptions of the C≡C and C≡N occur between 2100–2300 cm−1. In contrast, those arising from stretching absorptions of the C=O, C=N, and C=C occur between 1600–1850 cm−1.
The...

IR Spectrum

IR Spectrum

When infrared (IR) radiation passes through a molecule, the bonds stretch or bend by absorbing the radiation. This absorption creates the molecule's absorption spectrum, which is the plot of its percentage transmittance versus wavenumber.
Transmittance is defined as the ratio of the radiant power passing through a sample to that from the radiation's source. Multiplying the transmittance by 100 gives the percent transmittance (%T), which varies between 100% (no absorption) and 0% (complete...

IR Spectrometers

IR Spectrometers

There are two main infrared (IR) spectrophotometers: dispersive IR spectrometers and Fourier transform infrared (FTIR) spectrometers. In a dispersive IR spectrometer, a beam of infrared radiation produced by a hot wire is divided into two parallel equal-intensity beams using mirrors. One beam passes through the sample, while another is a reference beam. The beams then move through the monochromator, which separates the radiations into a continuous spectrum of different frequencies. The...

IR Spectrum Peak Intensity: Amount of IR-Active Bonds

IR Spectrum Peak Intensity: Amount of IR-Active Bonds

When infrared radiation is passed through a molecule, absorption occurs if the molecule's vibration leads to a substantial change in its bond dipole moment. Transitions between vibrational energy levels, typically corresponding to infrared frequencies (4000–400 cm−1), allow absorption if the vibration significantly alters the dipole moment, making the molecule infrared active. The molecular bonds have different stretching and bending vibrations, resulting in various peaks with varying...

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Related Experiment Video

Updated: Jun 14, 2026

Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy

Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy

Published on: June 27, 2014

Infrared spectroscopy for biopharmaceutical protein analysis.

Peter Christian Gross¹, Michael Zeppezauer

¹Symbiotec GmbH, Science Park Saar 1, D-66123 Saarbrücken, Germany.

Journal of Pharmaceutical and Biomedical Analysis

|April 13, 2010

Summary

A new mid-infrared transmission spectroscopy method offers high-throughput protein analysis for biopharmaceutical quality control. This technique provides quantitative analysis, identity and purity determination, and structural characterization of protein therapeutics.

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Last Updated: Jun 14, 2026

Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy

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Published on: June 27, 2014

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Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects

Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects

Published on: February 18, 2014

Area of Science:

Biotechnology
Analytical Chemistry
Spectroscopy

Background:

Current methods for protein therapeutic analysis, such as attenuated total reflection (ATR), are often surface-sensitive.
There is a need for robust, quantitative, and high-throughput analytical techniques for biopharmaceutical quality control.

Purpose of the Study:

To develop and validate a mid-infrared transmission spectroscopic method for protein therapeutics.
To compare the advantages of transmission spectroscopy with existing methods like ATR.
To demonstrate the method's utility in characterizing protein structure, purity, and interactions.

Main Methods:

Development and validation of a mid-infrared transmission spectroscopic method.
Application of Lambert-Beer's law for quantitative analysis.
Establishment of specific identity criteria for purity determination.
Characterization of protein structure, aggregation, and ligand binding in solution.

Main Results:

The transmission method demonstrated high sample throughput and compliance with Good Manufacturing Practice (GMP) requirements.
Advantages over ATR include quantitative analysis, identity and purity determination, structural characterization, and ligand binding studies.
Successful characterization of ONCOHIST drug substance and its interaction with phosphate ions was achieved.

Conclusions:

Mid-infrared transmission spectroscopy is a powerful and versatile tool for protein analysis in biotechnology.
This method supplements existing analytical techniques for biopharmaceutical quality control.
The developed method offers significant advantages for the comprehensive analysis of therapeutic proteins.