Chlorophyll, a crucial substance for photosynthesis in higher plants, refers to green pigments featuring a magnesium structure and a porphyrin backbone combined with nitrogen on four pyrrole rings. Most photosynthetic organisms contain some form of chlorophyll, with higher plants having two main types: chlorophyll a and chlorophyll b. Algae may also contain chlorophyll c and chlorophyll d, while cyanobacteria exclusively have chlorophyll a. Anaerobic photosynthetic bacteria contain bacteriochlorophyll.

The qualitative and quantitative analysis of chlorophyll and its various derivatives has found applications in various fields. In botany, it is utilized to study chlorophyll types and structures for plant classification. In oceanography, it is employed to analyze phytoplankton cellular abundance in the ocean to monitor marine red tide occurrences. In horticulture, it is used to investigate fruit and melon ripening mechanisms and preservation methods. In agronomy, it helps study photosynthesis mechanisms. In the food industry, it is used to analyze chlorophyll derivative additive contents and food color.

Currently, chlorophyll is widely used in chewing gum, hard candy, fruit juice, agar, pastry, and other foods (excluding acidic or calcium-containing foods, as they may cause precipitation). In the pharmaceutical industry, chlorophyll is known for its various functions, including improving constipation, lowering cholesterol, anti-aging, detoxification, anti-inflammation, deodorization, anti-cancer and anti-mutation properties, as well as anti-anemia and liver protection. It also aids in skin tissue regeneration, treating burns, chronic ulcers, inhibiting staphylococcus and streptococcus growth, and showing some effectiveness against gingivitis, halitosis, otitis media, among others.

Methods for Chlorophyll Extraction and Isolation

Chlorophyll extraction methods include acetone grinding, filtration, organic solvent immersion, ultrasonic extraction, microwave-assisted extraction, and supercritical flow extraction. Chlorophyll separation is primarily achieved through chromatography. Plant pigments mainly consist of fat-soluble carotenoids, lutein, chlorophyll, and water-soluble anthocyanins. During extraction, water-soluble anthocyanins can be filtered out based on similar phase solubility principles, followed by the separation of carotenoids, lutein, and chlorophylls using thin-layer chromatography, column chromatography, and high-performance liquid chromatography (HPLC).

Methods for Chlorophyll Analysis

Spectrophotometry

Berzelius initiated chlorophyll research in 1818. In 1941, Mackinney quantified chlorophylls a and b directly using spectrophotometry with 80% acetone as a solvent, enabling the determination of other chlorophylls and derivatives. In 1952, Richard and Thompson pioneered the simultaneous analysis of multiple pigments in oceanography using spectrophotometry. Parson and Strickland later revised the formulae for calculating chlorophylls and carotenoids to address contradictions in previous results. Spectrophotometry, while complex and time-consuming, is widely used for chlorophyll measurement due to its simplicity, accuracy, simultaneous multi-pigment determination, and suitability for processing numerous samples.

High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS)

HPLC-MS combines HPLC's high separation performance with MS's sensitivity and specificity. It does not require complete chromatographic separation between analytes and is unaffected by degradation products. Its multi-window detection capability allows for the simultaneous quantitative analysis of multiple components. This method, fast and accurate in determining various photosynthetic pigments, has become increasingly popular in recent years.

Reference

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.