Principles and methods for determination of chlorophyll content

First, the purpose of the experiment

1. Understand the distribution and properties of chlorophyll in plant tissues.

2. Master the principles and methods for determining chlorophyll content.

Second, the experimental principle

Chlorophyll is widely found in green plant tissues such as fruits and vegetables, and binds to proteins in plant cells to form chloroplasts. When plant cells die, chlorophyll is free, free chlorophyll is unstable, sensitive to light and heat; under acidic conditions, chlorophyll produces green-brown pheophytin, which can be hydrolyzed into bright green chlorophyll in dilute lye Acid salts as well as phytol and methanol. There are two kinds of chlorophyll in higher plants: chlorophyll a and b , both of which are easily soluble in ethanol, ether, acetone and chloroform.

There are various methods for determining the content of chlorophyll, among which are: 1. Atomic absorption spectrometry: The content of magnesium is determined by indirect determination of the content of chlorophyll. 2. Spectrophotometry: The spectrophotometer is used to determine the absorbance of the chlorophyll extract at the maximum absorption wavelength, and the content of each pigment in the extract can be calculated by Lambert-Beer law.

Chlorophyll a and chlorophyll b have maximum absorption at 645 nm and 663 nm, and the two absorption curves intersect at 652 nm. Thus the extract was measured at 645nm, 663nm absorbance at 652nm wavelength, and can calculate the amount of chlorophyll a, chlorophyll b, and total chlorophyll according to the empirical formula, respectively.

Third, the instrument, the instrument spectrophotometer starting materials and reagents, the top carrier electronic balance (the amount of feeling 0. 01g), a mortar, a brown flask, small funnel, quantitative filter paper, absorbent paper, wiping paper environment, dropper. Raw material Fresh ( or dried ) plant leaves Reagents 1. 96 % ethanol ( or 80 % acetone ) 2. Quartz sand 3. Calcium carbonate powder .

Fourth, the operation steps

Take fresh plant leaves ( or other green tissue ) or dry material, wipe the surface dirt and remove the midrib. Weigh 2g of the freshly cut sample , put it into the mortar, add a small amount of quartz sand and calcium carbonate powder and 3mL of 95 % ethanol, grind into a homogenate , add 10mL of ethanol , continue grinding until the tissue becomes white. Allow to stand for 3 to 5 minutes . Take one piece of filter paper in a funnel, wet it with ethanol, pour the extract into the funnel along the glass rod, and transfer the filtrate to a 100 mL brown volumetric flask; rinse the mortar, pestle and residue several times with a small amount of ethanol, and finally together with the residue. Pour into the funnel. The ethanol was sucked up with a dropper, and the chloroplast pigments on the filter paper were all washed into a volumetric flask. Until there is no green in the filter paper and residue. Finally, dilute to 100 mL with ethanol and shake well. The chloroplast pigment extract was measured for absorbance at wavelengths of 665 nm , 645 nm and 652 nm, with 95 % ethanol as a blank control.

V. Calculation

According to the empirical formula provided in the experimental principle, the contents of chlorophyll a , b and total chlorophyll in plant materials were calculated.