Biomaterials Tutorial

Attenuated Total Reflection Infrared Spectroscopy (ATR) and Infrared-Reflection-Absorption Spectroscopy (IRRAS)

Shrojal Desai and Maxi Boeckl
University of Washington Engineered Biomaterials

Infrared Spectroscopy Basics

Chemists always wonder about the chemical composition of any organic matter. One of the best ways to determine the composition of an organic compound is through the functional groups present in it, for example: carboxyl, amine, aldehyde, ketone, ester, halide, thionyl, etc. There are several spectroscopic techniques used for characterizing functional groups such as: UV-Visible, Nuclear Magnetic Resonance (NMR), and Mass Spectrometry. Infrared spectroscopy is one of these fundamental techniques. If you have seen a rainbow, you already know what infrared radiation is. A rainbow shows the optical (visible) part of the electromagnetic spectrum and the invisible infrared would be located just beyond the red side of the rainbow.

How can IR radiation identify functional groups?  Molecules are in constant motion, producing vibrations, such as bond stretching and bending. There are symmetric and asymmetric stretches and bends, and bending vibrations can be either in-plane or out-of-plane vibrations.

When the frequency of incoming infrared radiation matches the frequency of the vibrations, energy is absorbed by the vibrating bonds [1, 2].  This causes the bonds to stretch and bend a bit more.  In other words, the absorption of energy increases the amplitude of the vibration but does not change its frequency.  This change in amplitude can be seen in an IR spectrum as a peak at the frequency of the chemical bond that absorbed the energy (Fig. 1). For example, C-H bond in the alkanes demonstrate stretching vibration around 3000-2840 cm-1. Depending upon its vicinity from the neighboring group, the position of this bond vibration varies in the spectrum. Similarly, C-O, C-S, C-H, N-H, S-O, etc., demonstrate their unique vibration bands in the IR spectrum.

Figure 1.  Infrared spectrum in the transmission mode of N-Acetyl glycine, prepared as a KBr pellet. 

IR-Spectroscopy measurements are performed in solvents like chloroform, mineral oils, or for solid samples, as potassium bromide (KBr) pellets. However, for samples that cannot transmit light such as opaque films or surfaces of solid samples, it is impossible to perform this analysis in the usual absorbance mode.  This is when the ATR or IRRAS mode of IR spectroscopy can be used.

Attenuated Total Reflectance Infrared Spectroscopy (ATR-IR)

Harrick and coworkers [3] with their pioneering efforts, developed a technique called attenuated total reflectance spectroscopy, in which IR spectra of the thin films/ plates can be obtained from near surface region. In ATR-IR, the IR light passes through the optically denser crystal and reflects at the surface of the sample as shown in the figure. According to Maxwell’s theory [4], “when the propagation of light takes place through an optically thin, non absorbing medium, it forms a standing wave perpendicular to the total reflecting surface. If the sample absorbs a fraction of this radiation, the propagating wave interacts with the sample and (its energy or frequency) becomes attenuated, giving rise to a reflection spectra, very similar to the absorption spectra.” In other words, in order to determine the chemical composition of a surface, (i.e., surface of a leaf, petal or an unknown object) it can be done using the ATR accessory in the IR spectrophotometer. The infrared radiation is reflected from the surface of the sample, and the resultant spectrum reveals the functional groups present on the surface. Using this technique it is possible to determine the composition of a multilayered sample up to the sampling depth of ~ 5 microns. Ample literature [5-9]is now available on the characterization of thin film surface using ATR-FTIR.

Figure 2: Working of an ATR.

Infrared Reflection-Absorption Spectroscopy

A second surface technique, infrared reflection-absorption spectroscopy (IRRAS), also known as  grazing-angle infrared spectroscopy (GAIRS), can identify a wide range of functional groups present on the surface as well as their orientations relative to the reflective surface.  It is typically applied to thin films on highly reflective surfaces and is therefore, often applied to SAMs on gold surfaces. 

Greenler was the first to notice that absorption of infrared radiation was enhanced at high angles of incidence and only involves one polarization of the incident light [10-12]. For example, imagine two polarized sunglass lenses stacked together.  In one orientation, dispersed filtered light comes through due to “constructive interference” of the polarized light.  If one of the lenses is rotated 90°, no light comes through at all due to “destructive interference” of polarization. In IRRAS, the light that is measured consists of only the polarization which stems from constructive interference.  Figure 3 shows how the p- and s-polarized light interacts with the surface [10, 11]. The reflected parallel or p-polarized radiation has an amplitude which is almost twice that of the incident radiation, hn.   The components Ep and Ep’ constructively interact, i.e., the amplitudes are added together.  On the other hand, the incident and reflected vectors of the perpendicular or s-polarized light undergo a 180° phase shift relative to each other and cancel each other through destructive interference, resulting in zero absorbance. Thus, the only active vibrations which are observed consist of bonds vibrating in the direction normal (perpendicular) to the gold surface plane.  Theoretical considerations have determined that the resultant amplitude of the p-polarized component of electromagnetic radiation reaches a maximum at ~ 82°, which is called the grazing angle (q) [10, 11].Measurements are given as a spectrum similar to Figure 1.  In IRRAS spectra the individual peaks represent specific functional groups with bonds perpendicular to the reflective surface.

Figure 3.  Schematic representation of the interaction of infrared radiation with the reflective surface at glancing angle.

References:

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3. Harrick NJ. Internal reflection spectroscopy. New York: Interscience, 1967.
4. Maxwell Garnett JC. Philos. Trans. R. Soc. London Ser. A 203, 385(1904); 205, 237 (1906).
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11. Pemple ME. In Vickerman JC, editor. Surface analysis:  The principal techniques. New York: John Wiley & Sons Ltd.; 1997. p 267-312.
12. Greenler RG. J Chem Phys 1966; 44: 310-315.