An Introduction to IR and Raman Spectroscopy: Harnessing the Power of Light in Molecular Analysis

Dr Samuel Gourion-Arsiquaud

Director, Skin & BioSubstrates

Dr Philippa Cranwell

Technical Content Creator

Raman and IR spectroscopy harness the power of light to probe molecular structure. Although both have been used extensively within analytical chemistry laboratories for many years, their use within cosmetic science to probe penetration of actives, or how a product behaves in vivo, has been stilted due to technological limitations. However, a new generation of instrumentation has enabled IR and Raman measurements to be undertaken on biological samples, opening the door to wider application of these powerful techniques in formulation development and actives analysis. 

This short article will outline the principles behind IR and Raman spectroscopy alongside a brief discussion of where these techniques can be used in practice, and how they can provide considerable help with product development and claim substantiation. 

IR and Raman spectroscopy 101

Infrared (IR) and Raman are often referred to as ‘vibrational spectroscopies’, as both techniques collect data that relates to how molecular bonds vibrate when interacting with light. IR spectroscopy uses light energy across the entire infrared region of the electromagnetic spectrum, whereas Raman spectroscopy uses light either in the near-IR (NIR) or visible, or sometimes UV regions, often at 785 nm Figure 1. 

Figure 1: The part of the electromagnetic spectrum that IR and Raman use.

Image adapted from LibreTexts Chemistry

As noted above, when collecting IR data the whole IR region of the spectrum is used, whereas collection of Raman data uses a laser of specific monochromatic wavelength. This information is the key behind how these spectroscopies work: IR spectroscopy measures the absolute frequencies at which a sample absorbs radiation. By contrast, Raman spectroscopy measures the relative frequencies (relative to the frequency of the monochromatic laser used to excite the spectrum) at which a sample scatters radiation of one single frequency that it does not absorb, Figure 2. While the information these methods provide are similar, each provides specific information, making them complementary. 

Figure 2: Ilustration showing the key difference between interactions of a sample with IR and Raman spectroscopy

The Science Part: Infrared spectroscopy

IR spectroscopy uses the infrared (IR) region of the electromagnetic spectrum, which corresponds to wavelengths in the range 2 × 10-4 to 1 × 10-6 m. Within this window, a longer wavelength corresponds to a lower frequency (and therefore lower energy), and a shorter wavelength corresponds to a higher frequency (and therefore higher energy).  The energy associated with IR radiation, 4−40 kJ, causes bonds to ‘waggle’, stretch, scissor, rock, or bend, with the frequency of vibration (or stretching) of the bond directly proportional to the strength of the bond (and inversely proportional to the masses of the atoms in the bond), Figure 3. This means that bonds belonging to different functional groups will vibrate at different frequencies, with stronger bonds vibrating at higher frequency. In addition, different modes of vibration will occur at different frequencies; stretches tend to occur at higher frequency than bends as they require more energy. For example, O−H bonds undergo stretching between 3300 and 2500 cm−1, a higher frequency, but bend between 1450 and 1400 cm−1, a lower frequency. 

With IR the detector compares the frequencies of light entering the sample with the frequencies of light leaving the sample. The ‘missing’ frequencies correspond to energy absorption by the molecule (or by bonds in the molecule), allowing for identification of the presence of specific chemical entities. 

Figure 3: Image showing some modes of vibration of bonds upon interaction with infrared light.

The Science Part: Raman spectroscopy

Raman spectroscopy typically uses a laser in the near-IR (NIR) or visible region of the electromagnetic spectrum that emits a specific single wavelength of light. Rather than being based on radiation absorbance, Raman spectroscopy is based upon radiation scattering (in this case, light). 

In simple terms (and avoiding the physics!), when light hits the molecule, or a bond in the molecule, it is scattered, which changes the light’s energy. The difference in the energy of the light entering the sample and the energy of the light leaving the sample can be measured, giving a ‘Raman shift’. The Raman shift depends upon the frequency of natural vibrations within the bond or molecule, with the frequency of vibration affected by the mass of a molecule or atom (when considering a bond). 

The interaction of light with a bond can also be affected by the polarizability of the electron cloud. If a bond in a molecule has a large, diffuse, electron cloud it will scatter radiation more readily, giving a stronger signal. The scattered light is then collected at a detector, and the frequency difference between the incoming light and scattered light determined. In this way, Raman can differentiate between homonuclear bonds, for example C−C, C=C and C≡C, as there are large differences in electron cloud polarizability. 

Final science part: Jablonski diagram

There is one more thing to be aware of, which has thus far been glossed over: what actually happens when light is absorbed by the molecule? This requires some appreciation of physics: each bond has a low energy vibrational state (often called the ground state) and above this there are excited vibrational states of increasing energy. The vibrational states are defined by specific energy differences. 

There are three modes of energy absorption: 

• Rayleigh, or elastic scattering, where the light absorbed is the same energy as that emitted; 

• Stokes Raman scattering, or inelastic scattering, where the light absorbed is of higher energy than that emitted;

• anti-Stokes Raman scattering, or inelastic scattering, where the light absorbed is of lower energy than that emitted. 
  

These modes can be represented with a Jablonksi diagram, Figure 4. 

IR light does not have enough energy to significantly change the state of the bond, therefore irradiation with IR light only causes a slight increase in vibrational state of the bond; certainly not enough to be easily observable. IR therefore relies upon comparison on wavelengths of light entering the sample with those leaving the sample. If a wavelength is ‘missing’ it has been absorbed by a particular bond, i.e. the absolute frequencies at which a sample absorbs radiation, Figure 4 far left. 

In Raman spectroscopy, Stokes Raman scattering is the inelastic mode usually measured because at room temperature the molecules are normally in the lowest vibrational energy state possible, Figure 4, highlighted in blue. 

Figure 4: Jablonski diagram showing quantum differences in energy absorption/emission by IR, Rayleigh scattering, Stokes Raman scattering and anti-Stokes Raman scattering.

As noted earlier, Raman spectroscopy, rather than being based on absorbance of radiation, is based upon radiation scattering (in this case, light). In particular, Raman spectroscopy uses a laser with specific wavelength of light that, when it impacts with the electron cloud of a bond that is moving with specific molecular vibrations, can cause the light from the laser to be shifted to a different energy, or scattered. When the laser light hits the molecule or bond, the molecule or bond is excited to a ‘virtual energy state’; a very short-lived, unobservable quantum state. After excitation, the molecule or bond ‘falls back down’ to a ground state, which manifests as a shift in energy. This energy shift is given off as light of a specific frequency, which can be detected by the Raman detector. In this way, Raman spectroscopy irradiates the sample with one frequency of light and then measures the relative frequencies of scattered radiation, which correspond to different chemical entities. If a bond in a molecule has a large, diffuse electron cloud, it will scatter radiation more readily giving a stronger signal. 

That’s a lot of science. What does this actually mean in practice?

In simple terms, this means that molecular groups with non-polar bonds and an easily distorted electron cloud will be more intense with Raman, while molecular groups with polar bonds and a strong dipole will be more intense by FT-IR. For example, the water O–H stretch is significantly easier to observe by FT-IR when compared with Raman.

Figure 5: IR peaks highlighted in blue showing key bonds that can be monitored using IR spectroscopy, and green for Raman. Where a range is given, the exact location of the peak will depend upon the chemical environment in which the bond resides e.g. (C=O in an ester usually occurs between 1750 – 1730 cm–1, whereas in a ketone it is usually 1730 – 1700 cm–1).

Data adapted from Cranwell, Harwood and Moody, Experimental Organic Chemistry, 2017, 3rd Edition, Wiley and online.

Figure 6: Comparison of O–H stretch and bending modes.

FT-IR or Raman: do I need to choose?

Although both techniques can be used to successfully observe a range of chemical bonds, FT-IR is better when monitoring compounds containing chemical bonds with a strong dipole, for example O–H, N–H and C=O, whereas Raman is useful for studying bonds that are non-polar but have a polarizable electron cloud, for example C–H, C–O and C–N. This means that when using both techniques in tandem, a range of different molecules can be monitored, including water content, lipids and active ingredients e.g. caffeine. If possible, both FT-IR and Raman data should be collected, as both can help build a more complete picture. For example, if a sample contained caffeine and a client wanted to know how far the caffeine penetrated into the skin, FT-IR and Raman could be used to monitor for the presence different bonds across a transect of sample giving richer data, Figure 7. 

Figure 7: Molecule of caffeine. Bonds highlighted in blue are most suited to FT-IR spectroscopy as they have a strong dipole. Bonds highlighted in green (C–H) are most suited to Raman spectroscopy as they are easily polarised. The C=N bond, highlighted in yellow, can be easily monitored by both techniques.

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For further information about how TRI Princeton can use vibrational spectroscopy get in touch with us today.

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We would like to thank Matthew Almond, Emeritus Professor at the University of Reading, for his help and guidance when preparing this article.