How to Extract and Determine Chlorophyll in Laminaria Kelp is a vegetable with high nutritional value. Since seawater can use much less light energy than on land, to complete energy conversion, it is necessary to increase the content of chlorophyll involved in photosynthesis. Therefore, in general, the content of chlorophyll and other supplementary pigments in kelp is higher than that of terrestrial plants. Chlorophyll can be divided into chlorophyll a and chlorophyll b depending on its structure. Its basic structure is a magnesium-bound porphyrin ring. The current method for determining chlorophyll is mostly solvent extraction-visible spectrophotometry. Chlorophyll is separated by ultrasonic extraction, then magnesium ions are replaced by hydrogen ions, and the amount of magnesium can be measured by atomic absorption spectrophotometry to calculate the amount of magnesium in the kelp. Chlorophyll content, as well as the professional chlorophyll meter method. These methods are the most commonly used methods for the determination of chlorophyll. In the specific operations, we must make reasonable choices according to different needs. Chlorophyll extraction method: Weigh 4 g of crushed kelp in a beaker, add 30 mL of organic extractant, seal with plastic wrap, extract ultrasonic wave at room temperature of 200 W for 20 min, filter and collect the filtrate. Pipette 8mL of filtrate in a separatory funnel, add 4mL of petroleum ether and the same amount of saturated sodium chloride solution extraction, add 12mL of ultrapure water to wash the organic phase, repeated extraction twice to wash off the extraction solvent and inorganic salts, Combine the petroleum ether phase. In the petroleum ether phase, 15 mL of 0.9 mol/L hydrochloric acid was added for back-extraction for 15 min. The aqueous phase was separated, and the volume of ultra-pure water was set in a 50 mL volumetric flask. The mass concentration of magnesium in the solution was measured by an atomic absorption spectrophotometer. The measurement conditions were an air-acetylene flame, a magnesium wavelength of 285.2 nm, a slit width of 0.2 nm, a lamp current of 3 mA, and a burner height of 5.0 mm. For the determination of chlorophyll content in kelp by atomic absorption spectroscopy, see equation (1). Chlorophyll content/(mg/g)=[C×V1×899.3×V3]/V2×m×24×1000 (1). Where: C is the mass concentration of magnesium in the test solution from the standard curve / (μg/mL); m is the sample amount / g; V1 is the total volume of the mixed extract / mL; V2 is the removal of petroleum ether phase The volume/mL; V3 is the constant volume/mL of the backwashed aqueous phase; 899.3 is the average molar mass of chlorophyll a, chlorophyll b/(g/mol); 24 is the molar mass of magnesium/(g/mol). Ultrasonic extraction was used to extract chlorophyll from kelp. Atomic absorption spectrometry was used to determine the magnesium ions in the extracted chlorophyll, and the chlorophyll content in kelp was indirectly determined. The results showed that the optimum conditions for ultrasonic extraction of chlorophyll were as follows: a mixture of absolute ethanol and acetone with a volume ratio of 1:1 was used as the extractant, and the ratio of material to solution was 2:15 (g/mL). Ultrasound was used to select 200 W at room temperature. The power is extracted 2 times, 20 minutes each time. The optimum measurement conditions for back extraction: Back extraction with 15 mL of 0.9 mol/L hydrochloric acid for 15 min. Atomic absorption spectrometry detection limit of 0.008μg/mL, chlorophyll content in kelp 5.486mg/g, the relative standard deviation of 0.675%, the recovery rate of 97.7%. This method shows that atomic absorption spectrometry is less affected by interference and has higher precision, sensitivity, and accuracy.
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