FT-IR Spectroscopy Mini-Tutorial: Principles, Practice, and Applications Across Disciplines

Introduction and Relevance

Fourier transform infrared (FT-IR) spectroscopy has become one of the most widely used analytical methods in modern laboratories because virtually all molecules absorb some infrared (IR) radiation in characteristic ways (1-14). These absorption bands correspond to specific vibrational modes, allowing FT-IR to provide both qualitative and quantitative molecular information (1,12-14).

Compared with dispersive IR instruments, FT-IR spectrometers offer higher signal-to-noise ratios, better spectral resolution, faster data collection, and more reliable calibration transfer. These advantages have made FT-IR indispensable in materials science, chemistry, biopharmaceutical analysis, environmental monitoring, and food and polymer research (1,2,6,12-14).

This mini-tutorial provides a concise, practice-oriented overview of FT-IR spectroscopy, from fundamental principles to modern applications and experimental modes. Practical guidance is included to help readers select the appropriate measurement geometry, recognize potential sources of error, and apply FT-IR methods effectively in their daily analytical work.

Core Mini-Tutorial Contents

Principles and Definitions

FT-IR spectroscopy measures the absorption of infrared light by molecules undergoing vibrational transitions between quantized energy states. When IR radiation interacts with a sample, specific frequencies are absorbed that correspond to molecular bond vibrations, such as stretching, bending, or twisting of dipoles (12-14).

In a typical FT-IR instrument, a broadband IR source passes through an interferometer, most commonly of the Michelson design. A moving mirror produces a series of constructive and destructive interference patterns—an interferogram—that encodes all spectral frequencies simultaneously. The interferogram is then mathematically transformed by a fast Fourier transform (FFT) algorithm into an intensity-versus-wavenumber spectrum (12-14).

The main advantages of the Fourier transform approach include:

• Fellgett’s (multiplex) advantage: simultaneous measurement of all wavelengths improves signal-to-noise ratio.
• Jacquinot’s (throughput) advantage: fewer optical slits mean higher energy throughput.
• Connes’ advantage: high precision of wavelength calibration derived from an internal laser reference.

Infrared absorption depends on a change in dipole moment; therefore, polar bonds (C=O, O–H, N–H) are typically strong IR absorbers, whereas homonuclear diatomic molecules (N₂, O₂) are not.

How FT-IR Works in Practice

Modern FT-IR instruments can be configured for several sampling geometries: transmission, reflection, diffuse reflectance (DRIFTS), and attenuated total reflectance (ATR) (12-14).

1. Transmission: IR light passes through a thin film, gas cell, or KBr pellet. This mode is suitable for transparent samples but requires careful sample thickness control.
2. ATR (Attenuated Total Reflectance): The most popular modern technique. An internal reflection element (IRE) such as diamond, ZnSe, or Ge guides the IR beam through the sample interface. Penetration depth (~1–2 µm) enables direct analysis of solids, liquids, and gels without extensive preparation (6,9,12-14).
3. Diffuse Reflectance (DRIFTS): Scattered radiation from powders or rough surfaces is collected; excellent for soils, catalysts, or asphalt materials (5,8).
4. Specular Reflection and RAIRS: Used for thin films or monolayers on reflective substrates, particularly in surface and catalytic studies (12-14).
5. Photoacoustic (FT-IR-PAS) and Microspectroscopy (µ-FT-IR): Extend FT-IR to inhomogeneous, micro-scale, or non-transparent samples (6,9).

Proper background referencing is essential. A clean IRE or solvent reference should be recorded before each sample run to correct for atmospheric water vapor and CO₂ absorptions.

Application and Method Examples

1. Atmospheric and Environmental Monitoring

FT-IR has been extensively used to measure atmospheric gases such as CO₂, CH₄, and ozone. Open-path or extractive configurations allow in situ and remote sensing applications, using the sun or hot sources as IR emitters (1). These methods provide quantitative trace gas data but require careful calibration and correction for overlapping spectral bands and changing background conditions.

Micro-FT-IR (µ-FT-IR) has also become a leading method for detecting microplastics in environmental matrices such as water and sediment. Recent work standardized µ-FT-IR reflection and transmission modes, showing that diffuse reflection provides superior accuracy for small particles (9).

2. Food and Industrial Process Analysis

FT-IR enables rapid compositional analysis of oils, fats, and other food products. Al-Alawi and colleagues (2) developed a practical FT-IR method for free fatty acid (FFA) determination in edible oils. Using a potassium phthalimide reagent in 1-propanol, they achieved quantitative FFA analysis without titration. This approach illustrates the power of differential spectroscopy to remove matrix interferences, and it is automatable for high-throughput industrial use.

3. Materials Science and Polymers

Polymer scientists routinely use FT-IR to quantify crystallinity, detect oxidation, and monitor degradation. He and Inoue (10) demonstrated an FT-IR curve-fitting method for determining crystallinity in poly(ε-caprolactone), achieving agreement with conventional techniques. Similarly, Marsac and colleagues (5) used FT-IR to characterize oxidation in reclaimed asphalt binders, identifying challenges in harmonizing index calculations across laboratories.

In nanomaterials, FT-IR has proven invaluable for analyzing surface chemistry. Petit and Puskar (3) reviewed FT-IR methods for nanodiamond characterization, emphasizing the sensitivity of IR spectra to surface functional groups and the need for complementary methods for unambiguous assignment.

4. Biomedical and Pharmaceutical Applications

FT-IR spectroscopy has been widely adopted for protein structural analysis, biomedical materials, and diagnostics. Jiang and coauthors (11) qualified FT-IR for quantifying protein secondary structure, demonstrating >90 % reproducibility in replicate spectra and sensitivity to conformational changes due to pH or denaturants.

In materials modification and drug delivery, Kowalczuk and Pitucha (6) applied FT-IR-ATR to verify immobilization of active molecules in catheter matrices. FT-IR detected functional groups indicative of both covalent and non-covalent interactions, confirming successful drug incorporation. Such methods support the development of advanced biomaterials and implant coatings.

Wenning and Scherer (4) reviewed the use of FT-IR for microbial identification and strain typing. The ability to classify bacteria below the species level through spectral fingerprints has made FT-IR a valuable epidemiological and biotechnological tool.

5. Geochemical and Catalytic Studies

FT-IR spectroscopy provides insight into mineralogical composition and adsorption processes. Tkachenko and Niedzielski (8) summarized FT-IR methods for soils and sediments, highlighting the utility of DRIFT and synchrotron radiation (SR-FT-IR) for analyzing complex solid matrices.

In catalysis research, FT-IR is used to probe adsorbed species, identify active sites, and monitor reaction intermediates. Guerrero-Pérez and Patience (12-14) outlined the range of operando FT-IR techniques—including transmission, ATR, and reflection modes—demonstrating their application to heterogeneous catalysis, photocatalysis, and adsorption processes.

6. Biophysical and Photosynthetic Systems

Reaction-induced FT-IR difference spectroscopy allows the detection of structural changes in biomolecules with remarkable sensitivity. Berthomieu and Hienerwadel (7) described FT-IR’s ability to probe amino acid side-chain vibrations, using isotope labeling and site-directed mutagenesis to assign bands to specific residues. This method has elucidated mechanisms in photosynthetic complexes and other redox proteins.

Tips and Common Pitfalls

1. Sample Preparation: Inadequate sample thickness, uneven contact in ATR, or water contamination can distort spectra. Use dry nitrogen purging or desiccation to reduce atmospheric interference (1,12-14).
2. Spectral Resolution: Overly high resolution increases noise without adding useful information; typically, 4 cm⁻¹ suffices for most analyses (1,12-14).
3. Baseline and Background Correction: Apply proper baseline correction and reference spectra. Automated software should be verified manually when spectral features overlap (8,13,14).
4. Quantitative Analysis: When applying Beer–Lambert law, ensure linearity of absorbance with concentration, and use calibration models validated by independent standards (2,6,10,13,14).
5. Data Pre-Processing: Normalize spectra, remove baseline drift, and apply derivatives or chemometric tools (PCA, PLS) for multivariate interpretation, particularly in complex matrices (6,8,11,13,14).
6. Instrument Maintenance: Regularly clean ATR crystals, replace desiccants, and verify interferometer alignment using the built-in laser reference.
7. Method Validation: As shown in protein and polymer studies (10,11), FT-IR methods should be qualified for reproducibility, precision, and sensitivity before use in regulated environments.

Figures and Diagrams

For instructional purposes, the following figures would benefit readers when demonstrating FT-IR (13,14):

• Figure 1: Schematic of a Michelson interferometer and generation of an interferogram. This figure should depict the IR source, beam splitter, fixed and moving mirrors, detector, and the resulting interferogram.
• Figure 2: Comparison of common FT-IR sampling accessories (ATR, DRIFT, transmission, and reflection). Include diagrams showing beam paths and sample interfaces.
• Figure 3: Representative FT-IR spectra from selected applications. Examples: atmospheric gas absorption bands, FFA peaks in oils, protein amide I/II regions, and polymer carbonyl bands indicating oxidation or crystallinity.
• Figure 4: Workflow for quantitative FT-IR analysis. Steps: sample preparation → background acquisition → data collection → baseline correction → calibration/quantification → validation.

All figures should use labeled axes (wavenumber in cm⁻¹ versus absorbance) and concise captions to reinforce analytical interpretation.

Conclusion and Practical Takeaways

FT-IR spectroscopy continues to expand as a universal, non-destructive analytical method for molecular characterization. Its adaptability—from gases to solids, from microplastics to proteins—makes it indispensable in laboratories worldwide. The examples discussed demonstrate how FT-IR can:

• Monitor environmental pollutants (1,9),
• Support industrial quality control (2,5,10),
• Characterize nanomaterials and biomaterials (3,6,11), and
• Reveal fundamental biochemical mechanisms (7,12-14).

Successful application of FT-IR depends on understanding instrument configurations, optimizing spectral acquisition, and interpreting data critically. Proper background correction, calibration, and validation are essential for quantitative accuracy. As advanced techniques such as operando FT-IR, synchrotron-based microspectroscopy, and nano-FT-IR mature, the technique will continue to bridge the gap between molecular spectroscopy and real-world problem solving.

References

(1) Bacsik, Z.; Mink, J.; Keresztury, G. FTIR Spectroscopy of the Atmosphere. I. Principles and Methods. Appl. Spectrosc. Rev. 2004, 39(3), 295–363. DOI: 10.1081/ASR-200030192

(2) Al-Alawi, A.; van de Voort, F. R.; Sedman, J. New FTIR Method for the Determination of FFA in Oils. J. Am. Oil Chem. Soc. 2004, 81, 441–446. DOI: 10.1007/s11746-004-0920-9

(3) Petit, T.; Puskar, L. FTIR Spectroscopy of Nanodiamonds: Methods and Interpretation. Diamond Relat. Mater. 2018, 89, 52–66. DOI: 10.1016/j.diamond.2018.08.005

(4) Wenning, M.; Scherer, S. Identification of Microorganisms by FTIR Spectroscopy: Perspectives and Limitations of the Method. Appl. Microbiol. Biotechnol. 2013, 97, 7111–7120. DOI: 10.1007/s00253-013-5087-3

(5) Marsac, P.; Piérard, N.; Porot, L.; Van den Bergh, W.; Grenfell, J.; Mouillet, V.; Pouget, S.; Besamusca, J.; Farcas, F.; Gabet, T.; Hugener, M. Potential and Limits of FTIR Methods for Reclaimed Asphalt Characterisation. Mater. Struct. 2014, 47(8), 1273–1286. DOI: 10.1617/s11527-014-0248-0

(6) Kowalczuk, D.; Pitucha, M. Application of FTIR Method for the Assessment of Immobilization of Active Substances in the Matrix of Biomedical Materials. Materials 2019, 12(18), 2972. DOI: 10.3390/ma12182972

(7) Berthomieu, C.; Hienerwadel, R. Fourier Transform Infrared (FTIR) Spectroscopy. Photosynth. Res. 2009, 101(2), 157–170. DOI: 10.1007/s11120-009-9439-x

(8) Tkachenko, Y.; Niedzielski, P. FTIR as a Method for Qualitative Assessment of Solid Samples in Geochemical Research: A Review. Molecules 2022, 27(24), 8846. DOI: 10.3390/molecules27248846

(9) Rathore, C.; Saha, M.; Gupta, P.; Kumar, M.; Naik, A.; de Boer, J. Standardization of Micro-FTIR Methods and Applicability for the Detection and Identification of Microplastics in Environmental Matrices. Sci. Total Environ. 2023, 888, 164157. DOI: 10.1016/j.scitotenv.2023.164157

(10) He, Y.; Inoue, Y. Novel FTIR Method for Determining the Crystallinity of Poly(ε-Caprolactone). Polym. Int. 2000, 49(6), 623–626. DOI: 10.1002/1097-0126(200006)49:6<623::AID-PI435>3.0.CO;2-8

(11) Jiang, Y.; Li, C.; Nguyen, X.; Muzammil, S.; Towers, E.; Gabrielson, J.; Narhi, L. Qualification of FTIR Spectroscopic Method for Protein Secondary Structural Analysis. J. Pharm. Sci. 2011, 100(11), 4631–4641. DOI: 10.1002/jps.22686

(12) Guerrero-Pérez, M. O.; Patience, G. S. Experimental Methods in Chemical Engineering: Fourier Transform Infrared Spectroscopy—FTIR. Can. J. Chem. Eng. 2020, 98(1), 25–33. DOI: 10.1002/cjce.23664

(13) Handbook of Vibrational Spectroscopy; Chalmers, J. M.; Griffiths, P. R., Eds.; 1st ed.; John Wiley & Sons: Chichester, 2006. DOI: 10.1002/0470027320

(14) Workman, J., Jr. The Concise Handbook of Analytical Spectroscopy: Physical Foundations, Techniques, Instrumentation and Data Analysis; World Scientific Publishing–Imperial College Press: Singapore, 2016. DOI: 10.1142/8800.