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What to Keep in Mind When Analyzing Chlorophyll Samples using UV-Vis Spectrometry

By 03.27.2025

In some food industries, it can be important to determine the chlorophyll concentrations present in food products. Chlorophyll is the pigment responsible for the green color in plants and is a key component in photosynthesis.21 There are multiple different chlorophyll compounds found in nature, and food products by extension, with varied chemical structures.

Analyzing chlorophyll content in food products can help in assessing their nutritional value and detecting adulteration and can also serve as an indicator of quality and processing methods. For example, in canola oil, chlorophyll is an undesired contaminant as not only does it alter the color of the oil, but it can participate in unwanted reactions, lowering the overall quality5-7. Under these circumstances the determination of chlorophyll content can be vital.

One common analysis method is UV-Visible Absorption Spectroscopy.

UV-Vis spectroscopy is a technique used to measure how much ultraviolet (UV) and visible (Vis) light a substance absorbs.  In this method, a beam of UV or visible light is directed at a sample, which can be either a liquid or a solid. The light is either absorbed by or passes through the sample. By measuring the intensity of light which is able to pass through the sample and the light intensity without the sample present, the absorbance can be determined and is recorded as a function of wavelength. Different substances absorb light at different wavelengths, creating a unique absorption spectrum. By analyzing this spectrum, you can identify the substance and, through Beer’s law, determine its concentration in the sample.

We conducted a study of chlorophyll utilizing the UV-Vis technique.

The Chlorophyll Study

Chlorophyll exists in two primary forms, chlorophyll a and chlorophyll b, each absorbing light at different wavelengths. This unique property makes it possible to analyze and quantify chlorophyll content in food products, providing insight into the product’s quality, freshness, and processing history.

We used a UV-Visible spectrophotometer to analyze extracted chlorophyll from commercially available spinach samples, as well as analyze the chlorophyll content present in olive and canola oil samples. The resulting UV-Visible spectra were compared to chlorophyll standards measured on the instrument and further mathematical analyses were performed to determine the concentrations of chlorophyll a and b. For canola oil, the chlorophyll concentration was quantified following procedures outlined by the American Oil Chemists Society (AOCS).

UV-Visible Absorption Spectroscopy, Sample Prep, Processes, and Challenges

UV-Visible Absorption Spectroscopy is a powerful analytical technique used to measure the absorbance of light by chlorophyll at specific wavelengths. This method is preferred due to its accuracy, simplicity, and non-destructive nature. By measuring the absorbance at 665 nm and 645 nm, scientists can determine the concentrations of chlorophyll a and b, respectively.

The accuracy of chlorophyll analysis largely depends on proper sample preparation. Food samples are typically homogenized and extracted using solvents such as acetone or ethanol. The extract is then filtered to remove particulate matter, ensuring a clear solution for spectroscopic analysis.

The UV-Visible Spectroscopy procedure involves calibrating the spectrophotometer with standard chlorophyll solutions to establish a baseline. The absorbance of the prepared food sample is then measured, and the chlorophyll concentration is calculated using established equations. Calibration is crucial to account for any potential instrument variability and to ensure accurate results.

Because this technique is non-destructive, it does not alter the sample, allowing for further analysis if needed. It has high sensitivity, and can detect low concentrations of chlorophyll, making it suitable for a wide range of food products.  Additionally, the procedure is relatively quick, enabling high-throughput analysis in industrial settings.

While UV-Visible Absorption Spectroscopy is a robust method, some challenges need consideration:

  • Interferences: Other compounds in food samples may absorb light at similar wavelengths, potentially interfering with chlorophyll measurement. Proper sample preparation and method optimization can mitigate these interferences.
  • Solvent Selection: The choice of solvent for extraction can affect the efficiency and accuracy of chlorophyll analysis. Selecting the appropriate solvent and ensuring consistent extraction procedures are vital for reliable results.

Conclusion

You can read the results and discussion of the study of spinach and oil samples in our application note, Analysis of chlorophyll content in food products through UV-Visible absorption measurements. It outlines in detail, the preparation, instrumentation, the absorptivity spectra, wavelength maxima tables, results, and references. We concluded from those results that UV-Visible Absorption Spectroscopy provides valuable insights into product quality, processing methods, and development, helping to ensure that food products containing chlorophyll meet the highest standards.

As technology advances, this analytical method will continue to play a significant role in food quality assurance and innovation.

Additional Resources and References

References used above and in the application note:

  1. Björn, L.O.; Papageorgiou, G.C.; Blankship, R.E.; Govindjee, A Viewpoint: Why Chlorophyll a?, Photosynth. Res., 2009, 99, 85 – 98.
  2. Chen, M.; Blankenship, R.E., Expanding the Solar Spectrum Used by Photosynthesis, Trends Plant Sci., 2011, 16, 427 – 431.
  3. Miazek, K.; Ledakowicz, S., Chlorophyll Extraction from Leaves, Needles and Microalgae: A Kinetic Approach, Int. J. Agric. Biol., 2013, 6, 107 – 115.
  4. Daun, J.K, Spectrophotometric Analysis of Chlorophyll Pigments in Canola and Rapeseed Oils, Lipid Technol., 2012, 24, 134 – 136.
  5. Endo, Y.; Thorsteinson, C.T.; Daun, J.K., Characterization of Chlorophyll Pigments Present in Canola Seed, Meal and Oil, JAOCS, 1992, 69, 564 – 568.
  6. Mag, T.K.; Canola Oil Processing in Canada, JAOCS, 1983, 60, 380 – 384.
  7. Daun, J.K.; Thorsteinson, C.T., Determination of Chlorophyll Pigments in Crude and Degummed Canola Oils by HPLC and Spectrophotometry, JAOCS, 1989, 66, 1124 – 1128.
  8. Hsu, C.-Y.; Chao, P.-Y.; Hu, S.-P.; Yang, C.-M., The Antioxidant and Free Radical Scavenging Activities of Chlorophylls and Pheophytins, Food Sci. Nutr., 2013, 4, 1 – 8.
  9. American Oil Chemists Society. Official Method Cc 13d-55 Chlorophyll Pigments in Refined and Bleached Oils. In: Official Methods and Recommended Practices of the AOCS, 7th edition. Urbana, IL: AOCS.
  10. Porra, R.J.; Thompson, W.A.; Kriedemann, P.E., Determination of Accurate Extinction Coefficients and Simultaneous Equations for Assaying Chlorophylls a and b Extracted with Four Different Solvents: Verification of the Concentration of Chlorophyll Standards by Atomic Absorption Spectroscopy, Biochim. Biophys. Acta. Bioenerg. BBA-Bioenergetics, 1989, 975, 384 – 394.
  11. Hashimoto, T.; Chow, Y.-K.; Nakano, H., Hirao, K., Theoretical Study of the Q and B Bands of Free-Base, Magnesium, and Zinc Porphyrins, and their Derivatives, J. Phys. Chem. A, 1999, 103, 1894 – 1904.
  12. Croce, R.; Cinque, G.; Holzwarth, A.R.; Bassi, R., The Soret Absorption Properties of Carotenoids and Chlorophylls in Antenna Complexes of Higher Plants.
  13. Nigam, S.; Rutan, S., Principles and Applications of Solvatochromism, Appl. Spectrosc., 2001, 55, 362A – 370A.
  14. Khalyfa, A.; Kermasha; S., Alli, I., Extraction, Purification, and Characterization of Chlorophylls from Spinach Leaves, J. Agric. Food Chem., 1992, 40, 215 – 220.
  15. Domenici, V.; Ancora, D.; Cifelli, M.; Serani, A.; Veracini, C.A.; Zandomeneghi, M., Extraction of Pigment Information from Near-UV Vis Absorption Spectra of Extra Virgin Olive Oils, J. Agric. Food Chem., 2014, 62, 9317 – 9325.
  16. Scott, K.J., Detection and Measurement of Carotenoids by UV/VIS Spectrophotometry, Curr. Protoc. Food Anal. Chem., 1, 2001, F2 – 2.
  17. Alves, F.C.G.B.S.; Coqueiro, A.; Março, P.H.; Valderrama, P., Evaluation of Olive Oils from the Mediterranean Region by UV-Vis Spectroscopy and Independent Comonent Analysis, Food Chem., 2019, 273, 124 – 129.
  18. Gandul-Rojas, B.; Cepero, M.R.-L.; Minguez-Mosquera, M.I., Use of Chlorophyll and Carotenoid Pigments Composition to Determine Authenticity of Virgin Olive Oil, JAOCS, 2000, 77, 853 – 858.
  19. Minguez-Mosquera, M.I.; Rejano-Navarro, L.; Gandul-Rojas, B.; Sánchez-Gómez, A.H.; Garrido-Fernandez, J., Color-Pigment Correlation in Virgin Olive Oil, JAOCS, 1991, 68, 332 – 336.
  20. Niewiadomski, H.; Bratkowska, I.; Mossakowska, E., Content of Chlorophylls and Carotenes in Rapeseed Oil, JAOCS, 1965, 42, 731 – 734.
  21. National Geographic Education website, © 1996 – 2025 National Geographic Society, https://education.nationalgeographic.org/resource/chlorophyll/

 

 

 

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