Chapter 3

The detectives get familiar with Raman spectroscopy, compare it to NIR

🗂 Workshop overview: Following the successful conclusion of their first case, the detectives sit down for a quick introduction to other useful tools for future investigations. In their newest training, they enhance their knowledge of Raman spectroscopy and see how this technique complements the more commonly used NIR spectroscopy method for food testing.

Shallot Holmes and the food detectives are sitting comfortably on the sofa, an empty electronic whiteboard before them. After successfully closing a case with the help of extraction and Kjeldahl methods, Shallot Holmes wants to hold a workshop on another commonly used technique in food analytics: vibrational spectroscopy. Specifically, the goal of today’s workshop is to compare spectroscopy methods used in food testing.
Nancy Beef stands up nervously and takes the plastic pencil. She begins to outline the basic principles of vibrational spectroscopy for her colleagues. She takes a deep breath and begins to explain.

Vibrational spectroscopy

If you hit a molecule with energy at a frequency between 10,000 cm-1 and 100 cm-1, the energy will be absorbed by the molecule and converted into energy of molecular vibration. There are different types of molecular vibrations:

Stretching vibrations – occur when the interatomic distance between two atoms increases or decreases along the bond axis
Bending vibrations – occur when there is a change in the angle between two bonds of a common atom or there is movement of a group of atoms with respect to the remainder of the molecule

The frequency or wavelength of absorption at which a vibration occurs depends on several factors:
• The relative mass of the atoms
• The bond strength, or the force constants of the bonds
• The environment of the atoms

These factors vary for different groups of atoms, also known as functional groups. Hence, characteristic peaks form at or near the same frequency depending on the molecular structure. This is the premise for compound identification using vibrational spectroscopy.

Nancy Beef looks around the room. The other food detectives are following closely and nodding along. She says she would like to proceed by discussing two common techniques for vibrational spectroscopy: Raman spectroscopy and near-infrared (NIR) spectroscopy. Miss Mapple happily exclaims that she loves Ramen noodles. Nancy Beef laughs and corrects that she will be talking about Raman spectroscopy, not Ramen spectroscopy. She begins to describe the technique.
Raman spectroscopy

The Raman process for acquiring Raman spectra requires the following steps:

1. Irradiation of a sample with a monochromatic source, such as a laser. Here, the frequency, or energy or irradiation must be higher than the resonant vibrational frequencies of a molecule.

2. The incident photons cause a transient distortion of the electron distribution of molecular bonds, or a change in polarizability, which leads to photon absorption.

3. The absorption process lifts electrons to a virtual, non-quantized energy level, from which photons are re-emitted, or scattered, and detected.

In general, atoms in which electrons are far from the sphere of influence of the positively charged nucleus are more polarizable.
Raman radiation could be either elastically or inelastically scattered:
Stokes Scattering – inelastically scattered radiation is of lower frequency than the excitation frequency
Anti-Stokes Scattering – inelastic scattering occurs at a higher frequency than the excitation frequency
Rayleigh scattering – elastic scattering occurs when a photon is emitted with the same energy as the excitation source

Only inelastically scattered photons provide information regarding molecular vibrations.

Stokes emission is generally favored for data analysis due to higher signal intensities than anti-Stokes scattering. The more intense signal is a consequence of a greater population of electrons in the ground state relative to the excited state.
Raman spectra are recorded according to the wavenumber shift from the excitation frequency, which is equivalent to the vibrational frequency. The change in energy produced by the vibration is equivalent to that observed in the absorption if the bond is IR-active. Hence, the Raman frequency shift and the IR absorption frequency are identical.
The Raman shift is independent of the excitation wavelength. Rather, the intensity of a Raman band depends on:

– The polarizability of the molecule
– The source intensity
– The concentration of the active group

Raman intensity is, in fact, directly proportional to the concentration of the analyte.

Since she knows her colleagues have a better grasp of the other type of vibrational spectroscopy, NIR, Nancy Beef gives just a fleeting overview of the technique.

NIR spectroscopy

NIR uses light between 10,000 and 4000 cm-1 to excite molecular vibrations. The molecular bonds that are most responsive to excitations at this frequency are most often polar. The molecular vibrations change the bond’s dipole moment. Absorptions in the near-infrared region are less intense than those required for Raman spectra, which results in deeper penetration of source light into the material.
Nancy Beef proceeds to compare NIR and Raman spectroscopy, showing clearly why often the two methods are considered complementary to each other.

Similarities between Raman spectroscopy and NIR spectroscopy

• Both provide a structural fingerprint for molecule identification
• Both are non-destructive, enabling direct analysis of intact samples
• Both spectrometers provide measurements in seconds to a few minutes
• Both spectrometer types can be interfaced with probes to improve sample accessibility

Differences between Raman spectroscopy and NIR spectroscopy

• Vibrations involving polar bonds (C-O, N-O, O-H, etc.) are weak Raman scatterers, but strong NIR absorbers. Conversely, nonpolar bonds (C-C, C=C, C-H) are well observed in Raman spectra, but not in NIR spectra.
• Peaks in an NIR spectrum are broader than Raman peaks and tend to overlap . Raman spectra have sharper peaks or signals. Raman offers better selectivity in certain cases and is better suited for analysis of unknowns.
• Water is a strong absorber in NIR, but it is inactive in Raman. Raman analysis might be then beneficial when water is a major interference (e.g. monitoring of crystallization in aqueous solution). When water is an analyte of interest (ex. monitoring of a drying process), an NIR method is appropriate
• NIR is preferred for non-crystalline, hydrophilic molecules, which are often poor Raman-scatterers. Smaller, aromatic, heterocyclic molecules are better detected with Raman.
• Some Raman lasers may cause sample heating and chemical degradation. Laser excitation may also cause sample fluorescence or photobleaching, which impacts baseline effects. This is especially prominent for non-crystalline (amorphous) samples

Nancy Beef decides to wrap up the workshop by looking into use of the two techniques in food testing. She mentions that IR spectroscopy is more widely used than Raman spectroscopy most likely due to quantitative possibilities, and lower cost of the NIR systems. However, she points out that Raman spectroscopy brings its own advantages, such as study of aqueous systems, and these should not be ignored.

The five food detectives agree to make good use of vibrational spectroscopy, as a powerful multicomponent analysis tool, in future cases of investigating analytes in food.
The team decides to grab some Ramen noodles for dinner. The five investigators head out into the evening, excited for their next case.