Experiments for the determination of the physical and chemical properties of chlorophyll
Experiments for the determination of the physical and chemical properties of chlorophyll
Experiment to understand some of the main physical and chemical properties of chloroplast pigments.
Operation method
Experiments for the determination of the physical and chemical properties of chlorophyll
Principle
Chlorophyll is an ester of a dicarboxylic acid that can be saponified with a base to produce a salt that is soluble in water, which can be used to separate chlorophyll from carotenoids. Chlorophyll and carotenoids both have conjugated double bonds and exhibit certain absorption spectra in the visible region, which can be examined spectroscopically or measured accurately with a spectrophotometer. The excited state chlorophyll molecule into which chlorophyll is converted after absorbing light quanta is very unstable, and when it returns to the ground state, it can emit red light quanta, thus producing fluorescence. Chlorophyll is also chemically unstable and is easily damaged by bright light, especially when chlorophyll is separated from proteins. Chlorophyll molecule of magnesium can be replaced by H + and become brown to magnesium chlorophyll, the latter encounter Cu2 + can form a green copper chlorophyll, this chlorophyll in the light is not easy to be damaged, it is commonly used in this method to produce green succulent plant dip specimens.
Materials and Instruments
Spinach leaf Papaya leaf Move 1. Chloroplast pigment extraction For more product details, please visit Aladdin Scientific website.
95% alcohol KOH methanol solution Acetic acid Benzene Acetic acid copper powder Calcium carbonate Quartz sand
Balance Spectroscope Small electric stove Test tube Measuring cylinder Pipette Mantle Small beaker Funnel etc.
Weigh the fresh leaves 2g cut in a mortar, add a small amount of 95 ﹪ alcohol (or 80 ﹪ acetone) and a small amount of CaCO3 powder and quartz sand, grinding into a homogenate, and then add 95 ﹪ alcohol (or 80 ﹪ acetone) l0 ml diluted grinding, filtered through filter paper in a vial, filtrate that is, the chloroplast pigment extract.
2. Saponification of chlorophyll (separation of green and yellow pigments)
(1) Pipette about 5 ml of chloroplast pigment extract into a test tube, and then add 1.5 ml of about 20 % KOH methanol solution, shake well.
(2) A moment later, add 5 ml of benzene, shake well, and then slowly add 1 ~ 1.5 ml of distilled water along the wall of the test tube, gently mixed (do not shake vigorously), static test tube on the rack, you can see that the solution is gradually divided into two layers, the lower layer of dilute ethanol solution, which is dissolved saponification of chlorophyll a and b (as well as a small amount of lutein), the upper layer of benzene solution, which dissolved in yellow carotenoids and lutein.
3. Substitution of Mg2+ in the chlorophyll molecule by H+ and Cu2+
(1) Pipette about 5 ml of chloroplast pigment extract into a test tube, add a few drops of 30 % acetic acid, shake well and observe the change in color of the solution.
(2) When the solution turns brown, pour out half of it into another test tube, put in a few grains of copper acetate powder, heat it slightly and observe the change in the color of the solution, comparing it with the tube without copper acetate.
(2) Explain the above process of color change and list its reaction formula.
4. Fluorescence of chlorophyll
Take 5 ml of a relatively concentrated chlorophyll extract into a test tube and observe the transmitted and reflected light of the solution under direct light.
What is the difference in the color of the transmitted and reflected light? Explain why.
5. Destruction of chlorophyll by light
(1) Take two test tubes, add 5 ml of diluted chloroplast pigment extract to each, place one tube in direct light and the other in the dark, and observe the change in the color of the solution in the two tubes after 1 to 2 h.
(2) Take two chromatograms separated by paper chromatography in the above experiment I, put one under direct light and the other in the dark. After about 1 h, compare the color changes of each of the four pigments on the two chromatograms.
(3) Explain the above results.