The light energy utilized during photosynthesis in higher plants is absorbed by chloroplast pigments (photosynthetic pigments). Chloroplast pigments are composed of chlorophyll A, chlorophyll B, carotene and lutein.

The extraction, isolation and determination of chloroplast pigments is the first step in studying their properties and their roles in photosynthesis. The chlorophyll content of leaves is closely related to photosynthesis and is an important indicator of the physiological state of reversed leaves.

The chlorophyll content is often measured in plant photosynthetic, developmental, and resistance physiology. The chlorophyll content is also an important indicator to guide crop cultivation, production and selection of crop varieties.

Principle

Chlorophyll is insoluble in water but soluble in organic solvents and can be extracted by grinding or soaking in various organic solvents, such as acetone, ethanol or dimethyl sulfoxide.

Chlorophyll has a maximum absorption of light of a specific wavelength in a specific extraction solution, and the absorbance (also called optical density) of the chlorophyll solution at this wavelength is measured with a spectrophotometer, and then according to the absorption coefficient of chlorophyll at this wavelength. Calculate the chlorophyll content.

The determination of chlorophyll content by spectrophotometer is based on the Lambert-Beer law：

when a beam of monochromatic light passes through a solution, the absorbance of solution is proportional to the product of concentration of the solution and the thickness of the liquid layer.

Its mathematical expression is: A=K*b*c (where: A is the absorbance;

K is the absorption coefficient;

b is the thickness of the solution;

c is the concentration of the solution);

The acetone solution of chlorophyll a and b in the visible light range.

The maximum absorption peaks are located at 663 and 645 nm, respectively. The absorbance coefficients of chlorophyll a and b at 663nm (the absorbance when the solution thickness is 1cm and the chlorophyll concentration is g•L-1) are respectively and; the absorbance coefficients at 645nm are respectively and.

According to Lambert-Beer law, the absorbance of chlorophyll solution at 663nm and 645nm (A663 and A645) is related to chlorophyll a, b and total concentration (a+b) in the solution (Ca, Cb, Ca+b, the unit is g•L- 1), the following equations can express the relationship:

A663=+ (1)
A645=+ (2)

Solving equations (1) and (2) gives:

Ca= A663—A645 (3)
Cb= A645—A663 (4)
Ca + b = A645— A663 (5)

Materials, instruments and medicines for the experiment

1. Material: Plant green leaves.
   2. Instruments: spectrophotometer, cuvette, balance, mortar, filter paper, funnel, dropper, scissors, 50ml volumetric flask.

3. Experimental steps

1. Extraction of chlorophyll: Use washing powder to clean the oil stains and dust on the surface of the leaves.

After drying, accurately weigh the leaves of Jiulixiang with an electronic balance, cut them into pieces and put them in a mortar.

Add 2~3ml of 95% ethanol to the mortar, then a little CaCO3 and quartz sand, grind into a homogenate, then add 10ml of absolute ethanol, continue grinding until the tissue turns white, and stand still in the dark for 3~5min.

Take a piece of filter paper in the funnel, wet it with absolute ethanol, introduce the grinding liquid along the glass plate into the funnel and filter, rinse the mortar, pestle and filter residue with absolute ethanol several times, transfer the filtrate to a 50mL volumetric flask, and finally use Dilute the volume to 50 mL with water-ethanol, which is the extract.

• Determination of absorbance:
  Take a clean cuvette, be careful not to touch the glossy surface of the cuvette with your hands, wash with a small amount of pigment extraction solution for 2 to 3 times, and pay attention to making the cleaning solution contact all parts of the inner wall of the cuvette;

Then pour the pigment extraction solution into the cuvette, the height of the liquid level is about 4/5 of the height of the cuvette, suck up the solution sprinkled on the outside of the cuvette with filter paper (be careful not to wipe), and then use the lens cleaning paper Wipe dry. Place the cuvette on the cuvette holder of the instrument, taking care not to spill the solution into the instrument.
Place a cuvette containing absolute ethanol as a blank control in the first position. Adjust the instrument’s wavelength to 663 and 645 nm respectively, adjust the transmittance to 100% with absolute ethanol as the blank control, and measure the absorbance of the solution at the above three wavelengths respectively.
Each sample was measured in 3 replicates. Note that the transmittance should be adjusted to 100% with absolute ethanol every time the wavelength is switched.

4. Result calculation

1. Calculation of results: Substitute the absorbance measured at 663 and 645 nm into formulas (3), (4) and (5) in the above principle to calculate the concentrations of chlorophyll a, b and total concentration.

A645 absorbance value A663 absorbance value Chlorophyll a Chlorophyll b Total chlorophyll content

Gourmet before dilution

After 5-fold dilution
Purple-backed leaves

 Results Analysis and Experiment Summary

1. The total mass of the samples used in the chlorophyll extract is different. The weight of the sample of Jiulixiang taken is 1g, so the measured absorbance is greater than 1.

The calculation of this set of data will lead to a large error in the final result of the experiment. The chlorophyll extract of the species (Julisa) was first diluted five times and then measured, and the normal absorbance value was obtained.

2. Chlorophyll is insoluble in water but soluble in organic solvents. It can be extracted by grinding with various organic solvents such as ethanol, acetone or dimethyl sulfoxide or by soaking and extracting chlorophyll. The analytical grade used in this experiment is ethanol, and its effect is relatively low compared to the acetone-ethanol mixture.

3. Factors affecting the determination of chlorophyll content may include: time and place of sampling, selected materials, selected parts, whether quartz sand was put into the grinding to prevent chlorophyll from being damaged, whether the grinding was sufficient, and whether there was any avoidance during the preparation of the extract. Light treatment, etc.