Infrared (IR) spectroscopy measures the wavelength and the intensity of the radiation absorbed by a sample when IR light – 780-2500 nm – passes through it. This technique is extensively used in several fields, both in the pharmaceutical, chemical, or polymer industry and academia.
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The great appeal of IR spectroscopy relies on its high versatility and applicability, with the possibility to analyze solid and liquid (even gas) samples, and the instrumentation being relatively non-expensive and easy to use. Moreover, the analysis is non-destructive and only requires minimal preparation.
Although quantitative studies are possible, infrared spectroscopy offers mainly qualitative information, allowing for the identification of organic compounds and in particular the determination of specific functional groups.
Moreover, thanks to recent advances that have significantly enhanced the capacity of IR spectroscopy in analyzing various types of biological samples, there is a rising number of studies investigating applications in the screening and diagnosis of various diseases.
IR radiation interacts with the vibrational modes
In simple terms, every molecule vibrates in different ways (vibrational modes) depending on the number of atoms and the type of bonds involved. The frequencies associated with these molecular vibrations, and therefore the vibrational bond energies, fall in the infrared region.
IR spectrometers have a light source that generates a beam of infrared light that passes through the sample while a second beam passes through a reference cell. Part of the radiation is absorbed by the sample, resulting in a reduction of intensity, when particular frequencies correspond to the vibrational bond energies of the functional groups in the molecule.
The instrument measures the transmittance (T), which is the intensity of the light transmitted by the sample divided by the intensity of the light transmitted by the reference cell. Very commonly, instead of the transmittance, IR spectrometers report the absorbance (A), which is given by the log10 of the reciprocal of T; A = log10(1/T).
The plot of either T or A as a function of the wavelengths, or particularly the wavenumbers (which are reciprocals of wavelengths in cm-1) produces the IR spectrum. The absorption frequency of IR bands is the same as the frequency of the molecular vibration that caused the absorption.
Identifying the fingerprints of functional groups
With several bands ranging from 200 to 4000 cm-1, IR spectra can be very complex. However, useful structural information can be obtained by the analysis of characteristic vibrational bands whose position and intensity provide a fingerprint of the molecular structure.
The IR absorption bands are qualitatively classified as strong (s), medium (m), or weak (w). Polar bonds like O-H are associated with strong bands, while symmetrical bonds might not show any band at all. The presence or absence of many functional groups usually can be determined.
For example, aliphatic C-H vibrations usually appear between 3000 and 2800 cm-1, while C-H of alkenes or phenyl rings appear between 3200 and 3000 cm-1. Carbonyl (C=O) vibrations usually appear between 1800 and 1600 cm-1, while C-O in alcohol show bands in the region 1260-1050 cm-1.
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Sample handling and analysis
Among the key features of IR spectroscopy is the possibility to analyze samples in the gas, liquid, and solid-state. Cells with different shapes and sizes can be used depending on the sample type and they are generally made of IR-transmitting materials, such as glass or quartz.
Gas samples are analyzed in 5-10 cm long cylinders. These cells are sealed with vacuum-tight gaskets, with stopcocks at each end allowing for the cells to be filled and emptied by a gas handling system.
Liquids, if slightly viscous, can be simply squeezed between two IR-transmitting plates and analyzed as thin films, with a thickness of c.a. 0.01 mm. Non-viscous liquids are analyzed in suitable cells with a thickness ranging from 0.01 mm to 4 mm.
There are several possibilities for the analysis of solid samples. They can be dissolved in a solvent (i.e. chloroform) and analyzed as a solution. Most of the solvents absorb IR radiation and can cause interference, although, the problem can be circumvented by subtracting the reference spectrum of the pure solvent.
A typical approach for the analysis of solids is to grind a few milligrams of the sample in mineral oil (Nujol mull). The resulting paste is spread between two IR-transmitting windows. The mineral oil however can show several bands in the IR spectrum that can interfere with the interpretation.
Solids are also very commonly analyzed via potassium bromide (KBr) disks. The sample is finely ground and mixed with KBr powder. The mixture is placed under a mechanical press affording a transparent disk, which is then put into the spectrometer and run. Although KBr itself does not show particular interferences, it is very hygroscopic, therefore strong H2O bands can appear in the spectrum.
Very often polymers are analyzed as films prepared by melts or solutions. The sample dissolved in an appropriate solvent is dried on a plate, producing a film that can be either analyzed on the support cell (KBr) or stripped off an analyzed without support. The film thickness is a limitation since thick samples do not allow the light to pass through them.
ATR for the analysis of surfaces
With thick or opaque films that do not transmit light, or when the analysis of surfaces is needed, IR spectra can be obtained with a technique is known as Attenuated Total Reflectance-Infrared (ATR-IR) spectroscopy. ATR is a phenomenon observed when an IR beam travels from a medium of high refractive index (i.e. zinc selenide crystal) to a medium of low refractive index (sample).
At a certain angle of incidence, some of the IR radiation penetrates beyond the crystal and is absorbed by the sample (up to 5 μm), which translates into the IR spectrum of the sample. It is very convenient and simple to use, hence why is widely applied not only to analyze rubbery materials and thick polymer films but also for the analysis of food, protein/drug interactions, and biofilms on surfaces.
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