First, the principles of experimental methods
1, chlorophyll widely exists in green plant tissues such as fruits and vegetables, and combines with protein to form chloroplasts in plant cells. Plant cells release chlorophyll when they die. Chlorophyll is unstable and sensitive to light and heat.
2. Chlorophyll generates green-brown pheophytin under acidic conditions, which can hydrolyze Liang Cheng green chlorophyll, chlorophyllin and methanol in dilute alkali solution. There are two kinds of chlorophyll in higher plants: chlorophyll a and chlorophyll b, both of which are soluble in ethanol, ether, acetone and chloroform.
3. There are many methods to determine chlorophyll content, among which the main ones are: atomic absorption spectrometry, which indirectly calculates chlorophyll content by measuring magnesium content. Spectrophotometry: the absorption value of chlorophyll extract at the maximum absorption wavelength is determined by spectrophotometer, and the content of each pigment in the extract can be calculated by Lambert-Beer law.
4. Chlorophyll A and Chlorophyll B have maximum absorption at 645nm and 663nm, and the two absorption curves intersect at 652nm. Therefore, the absorption values of the extract at 645nm, 663nm and 652nm were determined, and the contents of chlorophyll a, chlorophyll b and total chlorophyll were calculated according to empirical formulas.
Second, the experimental steps
1. Take fresh plant leaves (or other green tissues) or dry materials, wipe off the dirt on the tissue surface, remove the midvein and cut into pieces. Weigh 2g of fresh cut samples, put them into a mortar, add a small amount of quartz sand, calcium carbonate powder and 3ml of 95% ethanol, and grind them into a uniform slurry, then add 10mL ethanol, and continue grinding until the tissues turn white. Let stand for 3-5 minutes.
2. Put 1 piece of filter paper into a funnel, moisten it with ethanol, pour the extract into the funnel along the glass rod, and the filtrate will flow into a 100mL brown volumetric flask; Wash the mortar, grinding rod and residue several times with a small amount of ethanol, and finally pour them into the funnel together with the residue.
3. Absorb ethanol with a dropper, and wash all the chloroplast pigments on the filter paper into a volumetric flask. Until there is no green in the filter paper and residue. Finally, adjust the volume to 100mL with ethanol and shake well.
4. With 95% ethanol as the blank control, the absorbance of chloroplast pigment extract was determined at 665nm, 645nm and 652nm.
Third, matters needing attention
1, light has a destructive effect on chlorophyll, so the experimental operation should be carried out in weak light and ground as soon as possible.
2. If the pigment extract is mixed with other substances and causes turbidity, it will affect the determination of absorbance, so it should be re-filtered or centrifuged.
3. Before determining the absorbance of pigment, the wavelength of spectrophotometer should be corrected according to the instrument description and standard chlorophyll A and B, otherwise the determination accuracy of chlorophyll content will be affected.
Fourth, others.
1, the extraction time of anhydrous ethanol is long, and the chlorophyll extraction of some materials, especially woody plants, is incomplete. The extraction of anhydrous acetone will cause turbidity and affect colorimetric determination. Therefore, the mixture of acetone and anhydrous ethanol is more suitable for extracting chlorophyll than 2: 1.
2. The traditional 80% acetone method should be used cautiously because of its high water content, poor chlorophyll stability, slow extraction speed and low measured value.