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Method for Separation and Detection of Chlorophyll

Method for Separation and Detection of Chlorophyll

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Chlorophyll is an important substance for photosynthesis in higher plants. It is a general term for green pigments with a magnesium structure and a porphyrin backbone combined with nitrogen on four pyrrole rings. Most photosynthetic organisms contain some form of chlorophyll. The two main types of chlorophyll are chlorophyll a and chlorophyll b in higher plants. In some algae there is also chlorophyll c and chlorophyll d, while cyanobacteria contain only chlorophyll a. Anaerobic photosynthetic bacteria contain bacteriochlorophyll.

The qualitative and quantitative analysis of chlorophyll and its various derivatives has been applied in various fields. In botany, it is used to study the type and structure of chlorophyll for plant classification. In oceanography, it is used to analyze the cellular abundance of phytoplankton in the ocean to monitor the occurrence of marine red tides. In horticulture, it is used to study the ripening mechanism and preservation methods of various fruits and melons. In agronomy, it is used to study the mechanism of photosynthesis. In food industry, it is used to analyze the content of chlorophyll derivative additives and the color of food.

At present, chlorophyll has been widely used in chewing gum, hard candy, fruit juice, agar, pastry and other foods (but not for acidic or calcium-containing foods, otherwise, it is easy to produce precipitation). In pharmaceutical industry chlorophyll has the functions of improving constipation, lowering cholesterol, anti-aging, detoxification and anti-inflammation, deodorization, anti-cancer and anti-mutation, anti-anemia and liver protection. It has regenerative effect on skin tissue, can be used to treat burns, chronic ulcers, inhibit the growth of staphylococcus and streptococcus, and has certain effect on gingivitis, halitosis, otitis media, etc.

Chlorophyll Extraction and Isolation Methods

Chlorophyll is extracted by the following methods: acetone grinding, filtration, organic solvent immersion, ultrasonic extraction, microwave-assisted extraction and supercritical flow extraction.

The separation of chlorophylls is currently mostly done by chromatography. The pigments in plants mainly include fat-soluble carotenoids, lutein, chlorophyll and water-soluble anthocyanins. In the extraction, the water-soluble anthocyanins can be filtered out using the principle of similar phase solubility, and then carotenoids, lutein and chlorophylls can be separated using thin-layer chromatography, column chromatography and high-performance liquid chromatography (HPLC).

Chlorophyll Analysis Methods

  • Spectrophotometry

In 1818, Berzelius started his research on chlorophyll aspects. In 1941, Mackinney quantified chlorophylls a and b directly by spectrophotometry using 80% acetone as solvent, while other chlorophylls and other derivatives could also be determined. In 1952 Richard and Thompson used spectrophotometry for the first time for the simultaneous analysis of multiple pigments in oceanography.

In 1963 Parson and Strickland considered many contradictions in the results of Richard and Thompson and revised the formulae for calculating chlorophylls and carotenoids. This method is simple and convenient, highly accurate, allows simultaneous determination of multiple pigments, and facilitates the processing of large numbers of samples. Spectrophotometry is one of the most widely used methods to measure chlorophyll, but the operation process is complicated and the extraction time is long.

  • High performance liquid chromatography-mass spectrometry (HPLC-MS)

HPLC-MS combines the high separation performance of HPLC with the high sensitivity and specificity of MS. It does not require complete chromatographic separation between analytes and can be unaffected by their degradation products. Its multi-window detection capability allows for the simultaneous quantitative analysis of multiple components. The method is fast and can accurately determine the content of various photosynthetic pigments. With the continuous development in the last decade, it is gradually becoming one of the widely used analytical methods nowadays.

Method for Separation and Detection of ChlorophyllMass Spectrometry of non-allomerized chlorophylls a and b derivatives (Viera et al., 2018)


  1. Viera, I., Roca, M., & Perez-Galvez, A. (2018). Mass Spectrometry of Non-allomerized Chlorophylls a and b Derivatives from Plants. Current Organic Chemistry, 22(9), 842-876.

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