Chlorophyll determination methods
Chlorophyll widely exist in fruits and vegetables and other higher green plants, combined with proteins into chloroplasts. Higher plant chlorophyll has two kinds: chlorophyll a and chlorophyll B. These two kinds of chlorophyll soluble in ethanol, ethyl ether, acetone and other organic compounds. Chlorophyll is the necessary factor of photosynthesis of green plants during photosynthesis, to absorb and light transmission effect. Chlorophyll a formula C40H70O5N4Mg, chlorophyll a molecular structure composed of 4 pyrrole ring by 4 methylene ( = CH ) connected to form a ring structure, called nitrogen determination apparatus porphyria ( ring with a side chain ). The porphyrin ring central with 1 mg atoms, and a cyclopentanone ( V ), the ring IV on propionic acid is phytol ( C20H39OH ), after saponification esterification formed with water soluble potassium. In an acidic environment, the porphyrin rings in the magnesium can be replaced by H, called pheophytin, brown, when using copper or zinc to replace H, its color and turns green, the pigment is stable under light, do not fade, no acid damage, dipping plant specimen is saved, by using this property.
Using spectrophotometer determination of chlorophyll a, chlorophyll a is the most common measuring method. Using spectrophotometer determination of chlorophyll extraction solution, first in the maximum absorption wavelength of light absorption value, chlorophyll a in 645nm and 665nm at maximum absorption value. Then using Lambert – Bill’s law to calculate the pigment content in the extract of. Lambert – Bill law mathematical expressions for the A = LG ( 1 / T ) = Kbc, where A is the absorbance; T transmittance, projecting light intensity than the intensity of the incident light; C as light absorbing substance concentration; B absorption layer thickness.
Determination of chlorophyll a
First to introduce our in the determination process to use instruments: spectrophotometer, content of chlorophyll meter, balance, mortar, brown flask, portable chlorophyll meter, small funnel, quantitative filter paper, absorbent paper, wiping the exit paper, dropper; for some fresh leaf; ready to 96% ethanol ( or 80% acetone ), quartz sand and calcium carbonate powder.
Then the specific operation as follows : fresh leaves of the plant ( or other green tissue ) or dry materials, clean the surface dirt removing tissue, midvein shear. With shredded fresh samples 2G, into the mortar, plus a small amount of quartz sand and calcium carbonate powder and 3mL95% ethanol, research into all the oar, plus ethanol 10mL, continued to grind to tissue white. Static 3 ~ 5min. Take 1 pieces of a filter funnel, wet with ethanol, along the glass rod to extract into a funnel, a filtrate stream to 100mL Brown volumetric flask; with a small amount of ethanol flushing, pestle and mortar residue several times, finally together with the residue into the funnel. Dropper from ethanol, filter paper on chloroplast pigment all washed into the volumetric flask. Until the filter paper and residue in green. The final ethanol volume to 100mL, shake. Take the chloroplast pigment extract at the wavelength of 665nm, 645nm and 652nm measured absorbance, with 95% ethanol as blank control.