Techniques - Spectroscopy

Varieties of Spectroscopy

The frequencies of electromagnetic radiation wavelengths used for food authenticity testing applications are Infra-red (IR, approx. 700 – 4000nm), Near Infra-red (NIR, approx. 780 – 2500nm), UV-Visible (approx. 10 – 700nm). The wavelength range chosen will try to be appropriate to determine certain molecular markers in the sample molecules.

Image by the authors

Reference spectra are taken from authentic samples. If the test sample spectrum deviates (usually following chemometric analysis,) it may indicate either adulteration or substitution.

Infra-Red (IR) Spectroscopy

Infra-red spectra show the vibrational absorptions of different molecular bonds. Different types of bonds, in different positions within the molecular structure, give different characteristic peaks.  The entire spectrum therefore gives a “fingerprint” of the molecule.Infra-red spectrometers can be miniaturised and used in-situ for rapid screening and testing

Near Infra-Red (NIR) Spectroscopy

NIR spectroscopy is very similar to IR spectroscopy except that it uses a narrower section of the IR spectrum i.e., 1250-4000cm^-1 (800-2500nm). This results in the peaks becoming broader than those in IR spectra, and chemometric analysis is essential. Again, it can be used in-situ there are a variety of commercial hand-held NIR spectrometers.  

Fourier-Transform Infra-Red (FT-IR) Spectroscopy

FTIR differs from single beam infra-red spectroscopy in that it relies on interference of various frequencies of light to collect a spectrum. The spectrometer consists of a source, beam splitter, two mirrors, a laser and a detector; the beam splitter and mirrors are collectively called the interferometer.

The IR light from the source strikes the beam splitter, which produces two beams of roughly the same intensity. One beam strikes a fixed mirror and returns, while the second strikes a moving mirror. When the two beams are combined by the beam splitter, this gives the interference infra-red beam.

The combined beam is then passed through the sample, where some energy is absorbed, and some is transmitted. The detector records the subsequent intensity. This signal is and processed using a computer. The untangling of the frequencies into a spectrum is done by the Fourier transform algorithm..  

A background single beam spectrum is collected without a sample. The ratio of these two spectra gives the sharp spectrum, which can then permit interpretation of the specific stretching and rotating atomic frequencies.

FTIR Images by LibreTexts Chemistry, Howard University,, licensed under Creative Common Attribution

 UV-Visible Spectroscopy

UV-Vis spectroscopy measures the amount of discrete wavelengths of UV or visible light that are absorbed by or transmitted through a sample in comparison to a reference or blank sample. Light energy is inversely proportional to its wavelength; thus, shorter wavelengths of light carry more energy and longer wavelengths carry less energy. Hence although IR spectra involve absorption by the stretching and rotation of atoms in a molecule, UV-Vis absorption involves promotion of electrons in a molecule to a higher energy state. UV-Vis spectrometers normally use light with a range of wavelengths from 150/200nm to 700/750nm, (the visible light range is 380-780nm).

The technique can be used quantitatively by measuring the size of absorption peaks.

Raman Spectroscopy

Raman spectroscopy uses intense monochromatic light from a laser to probe the chemical bonds in a sample, generating a spectrum that acts as a fingerprint, which can be used to characterise or identify the compounds in a sample. A visible light wavelengh is usually chosen for food analysis.  The high intensity laser light source shines on the sample, which interacts with molecular vibrations, or other excitations in the system, and causes the inelastic scattering of photons, known as Raman scattering. Three types of scattering are produced

• Stokes Raman scattering, which is the scattered light used in Raman spectroscopy with wavelengths greater than the laser light
• Rayleigh scattering, which is the strongest but with wavelengths equal to the laser, and
• Anti-Stokes scattering, with wavelengths less than the laser.

Stokes Raman scattering is when the molecule gains energy from the photon during the scattering (excited to a higher vibrational level) then the scattered photon loses energy, and its wavelength increases. On the other hand, Anti-Stokes Raman scattering is where the molecule loses energy by relaxing to a lower vibrational level, and  the scattered photon gains the corresponding energy and its wavelength decreases. Stokes Raman scattering is always more intense than the anti-Stokes and for this reason, it is nearly always the Stokes Raman scattering that is measured in Raman spectroscopy. Even so, it is still relatively weak, which is why an intense laser light, and an optimised detector are required to give the spectrum.

Fluorescence Spectroscopy

Fluorescence spectroscopy is based on absorption of photons from a visual or UV light  source, which excite the molecules in the sample from a ground state to an excited electronic state. When the molecule returns back to its ground state, the resulting energy is emitted as a photon, which causes the molecule to fluoresce. The intensities and frequencies of these photons are detected and analysed to give a spectrum, and this can be used to authenticate the sample. Like other forms of spectroscopy, fluorescence spectroscopy gives quantitative results if there has been a quantitative calibration with authentic standards.

There are multiple published applications for food authenticity testing, across a wide range of sample types and situations.  Some recent examples that have been featured on FAN blogs:

Infra-red Spectroscopy

Detecting cow’s milk in goat’s milk. It identifies β-carotene as a marker for cow’s milk, which is absent in goat’s milk.

IR spectroscopy (hand-held) has been used to authenticate oregano and detect adulteration with olive or cistus leaves. A collaborative trial was held to test 34 different IR spectrometers, which gave good results when all were calibrated in a standardised way

Near Infra-red Spectrospopy

Distinguishing between two varieties of cinnamon - Cinnamon verum (the true cinnamon) with the lower priced Cinnamon cassia

A portable NIR spectrometer was calibrated and used to differentiate between Basmati rice varieties and their potential adulterants; six GI protected rice varieties from specific regions of China; various qualities of rice in Ghana and Vietnam; as well locally produced and imported rice in Ghana.

NIR has been used to authenticate black pepper and garlic

FT-IR Spectroscopy

FTIR with chemometric analysis was also used to determine cow’s milk adulteration of goat’s milk by β-carotene determination. It has been used as a screening method for sucrose adulteration of milk, lard adulteration in butter, garlic adulteration, and pork in meat products.

UV-Visible Spectroscopy

The technique is best suited for coloured components and hence analysis of wines, oils and beverages is a particular important use.  An example is authenticating Extra Virgin Olive Oil by checking for the spectral contribution from chlorophyl, the main pigment.

Images by PhysicsOpenLab.org,, licensed under Creative Common Attribution

Raman Spectroscopy

Rapid distinction between beef, lamb and venison. Raman has also been used for detecting lard adulteration of butter, authenticating honey from Argentina and Romania, authenticating single malt whisky, determining fish species, and detecting common wheat adulteration of durum wheat.

Fluorescence Spectroscopy

This has been used to authenticate Manuka honey, authenticate species in fish and meat products, authenticate cinnamon, and walnut oil

Spectroscopic equipment is relatively cheap and accessible (compared to other high-end laboratory instrumentation), and can be suitable for portable and in-line applications.  (FT-IR is an exception). Specific varieties have specific strengths.

Care must be taken to prevent light scattering which causes inaccuracies.

Spectroscopy is difficult to use for determining molecular structure or composition when dealing with complex samples such as raw ingredients where there are many natural compounds present.

It is important that calibration of spectrometers is done in a standardised way, which covers the range of natural variables of the raw ingredients or foods being monitored.

Substance Identification ("Qualitative Analysis")

Spectroscopy will rarely give a definitive identification.  It is best suited as a screening tool.  As with all methods that rely on statistical matching against a "fingerprint" of authentic samples, the key is in how the authentic database has been constructed. Comparison of spectra of authentic samples and test samples will only work if the test sample is supposed to be the same as the calibrating authentic sample i.e., the authentic calibration has to cover the full range of food materials that will be tested and all natural variation, for example from season to season.  The pre-treatment of reference data (e.g. the statistics used for normalisation) is also key.

"Quantitative" Analysis

The amount of a substance (or type of food) can be quantified using spectral intensity if the instrument is calibrated with reference samples of known quantitative composition.  Again, the calibration is key.