Chlorophyll is a green pigment found in higher plants and all other photosynthetic organisms. Chlorophyll A and chlorophyll B are soluble in ethanol, ether and acetone and other solvents, insoluble in water and petroleum ether, therefore, can use polar solvents such as acetone, methanol, ethanol, ethyl acetate and so on to extract chlorophyll.
Chlorophyll extraction process:
Chlorophyll extraction is prepared in a semi-dark room at 25 ° C. The extraction steps are as follows:
(1) Take 1000 grams of fresh green leaves and crush them in a Wechsler.
(2) Put the crushed 1000 grams of green leaves into acetone with a small amount of calcium carbonate (temperature 20℃) for extraction, until the leaf fragments are colorless after filtering and cleaning.
(3) Put the filtered acetone extract into a funnel containing 1 liter of petroleum ether and 100mL of acetone, and then gently rotate, while adding distilled water until stratified. Most of the acetone and water-soluble impurities in the water layer are discarded, leaving only petroleum ether solution.
(4) After the petroleum ether solution is purified again with distilled water, it is cleaned more than 5 times with 200ml80% methanol solution containing petroleum ether and 0.01g oxalic acid, and finally yellow-green suspension is obtained.
(5) Dry the suspension with anhydrous sodium sulfate, and infiltrate it into a column made of 3cm thick sucrose powder. Then clean the precipitated pigments to remove carotenoids and make them only contain natural chlorophyll.
(6) Sucrose column containing natural chlorophyll is divided into two layers, the green layer has 4-10mm chlorophyll B layer, the other blue layer is 2-6mm chlorophyll A layer.
(7) The part located in the middle of the blue layer (about half of the blue layer) is put into ether, the suspension is filtered and extracted, cleaned with distilled water, dried with sodium sulfate, and filtered with utensils to obtain chlorophyll A.
(8) Remove the middle part of the green layer in (6), and quickly put it into the ether for filtering and washing to make chlorophyll B ether solution.
How to separate during extraction?
Chromatography is an important method for separation, purification and identification of organic compounds, especially in microanalysis is widely used. Pigments in fruits and vegetables mainly include fat-soluble carotene, lutein, chlorophyll and water-soluble anthocyanin. In extraction experiments, we can make use of the principle of similar miscibility water-soluble anthocyanin filtering, which in turn can make use of thin-layer chromatography, column chromatography, high performance liquid chromatography (HPLC) to separation of beta-carotene, lutein and chlorophyll, weakened due to the polarity of the three types of pigment in turn, can be appropriately choose single organic solvent or different ratio of mixed solvent as expansion agent and elution agent, The optimal separation conditions were determined.
