The absorption peak for quantitative analysis must be stable and also have strong absorption. In addition, the problem of interference needs to be considered. There are many methods of quantitative analysis concerning chlorophyll, so this absorption peak is generally not considered when other sensitive and convenient methods of quantitative analysis are available.

If there are a variety of light-absorbing substances in the solution, the total absorbance of this mixture at a certain wavelength is equal to the sum of the absorbance of each component at the corresponding wavelength, which is the summation of absorbance. This experiment to determine the content of chlorophyll a, chlorophyll b and carotenoids in the extract, only need to determine the extract at three specific wavelengths.

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Methods of Chlorophyll Determination:

The chlorophyll content of the Determination methods are mainly ultraviolet spectrophotometry, fluorescence analysis, in vivo chlorophyll meter method, photoacoustic spectrometry and high performance liquid chromatography. However, the most widely used method at present is the spectrophotometric method.

The absorption spectrum of chlorophyll extract shows that there are two strong absorption peaks, respectively, in the red and blue-violet regions, and the absorption spectra of chlorophyll solutions obtained from different extraction solvents and raw materials are relatively similar.

The maximum absorption peaks of chlorophyll a and chlorophyll b were near 663 nm and 645 nm in the red region, and near 429 nm and 453 nm in the blue-violet region, respectively. Due to the different extraction solvents and raw materials, the maximum absorption values obtained after spectral scanning of chlorophyll extracts may have a small range of fluctuation.

High-performance liquid chromatography (HPLC) is highly accurate and effective for quantitative detection of chlorophyll content. Methanol and acetone were used as the mobile phase at a volume ratio of 80:20, while glacial acetic acid with a mass fraction of 0.1% was added to the mobile phase at a flow rate of 1.0 mL/min. The quantification was carried out by utilizing the chromatographic peak area of each pigment, and the quantification of chlorophyll a and chlorophyll b could be obtained by the working curve by the external standard method.

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