Determination of chlorophyll content spectrophotometrically

Summary

Determination of chlorophyll content can be used to: (1) determine the photosynthetic capacity, i.e., health, of the plant; (2) as data for research to improve plant viability; and (3) to understand the distribution and nature of chlorophyll in plant tissues.

Operation method

spectrophotometry

Principle

Chlorophyll is widely present in green plant tissues such as fruits and vegetables, and combines with proteins in plant cells to form chloroplasts. When the plant cell death, chlorophyll is free, free chlorophyll is very unstable, light, heat sensitive; in acidic conditions chlorophyll generated green-brown demagnesium chlorophyll, in dilute alkaline solution can be hydrolyzed into bright green chlorophyllate, as well as chlorophyll alcohol and methanol. There are two kinds of chlorophyll in higher plants: chlorophyll a and b, both are soluble in ethanol, ether, acetone and chloroform. Chlorophyll content determination method has a variety of methods, the main ones are: 1. Atomic absorption spectrometry: through the determination of magnesium content, and then indirectly calculate the content of chlorophyll. 2. Spectrophotometry: the use of spectrophotometer to determine the chlorophyll extract in the maximum absorption wavelength of the absorbance value, can be calculated using Lambert-Beer law of the extraction of the content of the pigment. Chlorophyll a and chlorophyll b have maximum absorption at 645nm and 663nm, and the two absorption curves intersect at 652nm. Therefore, the absorbance values of the extracts at 645 nm, 663 nm and 652 nm were determined, and the contents of chlorophyll a, chlorophyll b and total chlorophyll were calculated according to the empirical formula.

Materials and Instruments

Plant material
96% ethanol acetone quartz sand calcium carbonate powder
Spectrophotometer Electronic top-loading balance Mason jar Brown volumetric flask Quantitative filter paper Absorbent paper Boundary paper Dropper funnel

Move

1. Take fresh plant leaves (or other green tissues) or dry materials, wipe the surface dirt, remove the midrib and cut. Weigh 2g of freshly cut samples, put them into a mortar, add a small amount of quartz sand and calcium carbonate powder and 3mL of 95% ethanol, grind them into a homogeneous slurry, then add 10mL of ethanol, and continue to grind until the tissue turns white. Let stand for 3--5min.

2. Take 1 piece of filter paper in the funnel, wet with ethanol, along the glass rod to pour the extract into the funnel, the filtrate flow to 100mL brown volumetric flask; with a small amount of ethanol rinse mantle, research stick and residue for several times, and finally together with the residue poured into the funnel.

3. Use a dropper to suck up the ethanol and wash all the chloroplast pigments on the filter paper into the volumetric flask. Until there is no green color in the filter paper and residue. Finally, the volume was fixed with ethanol to 100mL and shaken well.

4. Take the extract of chloroplast pigment and measure the absorbance at 665nm, 645nm and 652nm with 95% ethanol as blank control.

Caveat

1. Light has a destructive effect on chlorophyll, the experimental operation should be carried out in low light, and should try to grind in a short time.

2. If the pigment extract is mixed with other substances and caused turbidity, it will affect the determination of absorbance, and should be re-filtered or centrifuged.

3. Before the determination of absorbance of pigment, the wavelength of spectrophotometer should be corrected according to the instructions of the instrument and the standard chlorophyll a, b, otherwise it will affect the accuracy of the determination of chlorophyll content.

Common Problems

1. Anhydrous ethanol extraction time is longer and less complete for some materials, especially the chlorophyll extraction of woody plants. Anhydrous acetone extraction will appear turbidity phenomenon, affecting the colorimetric determination. Therefore, the use of acetone and anhydrous ethanol mixed extraction of chlorophyll is more effective, the ratio of 2:1 effect is better.

2. 80% of the traditional acetone method due to high water content, resulting in poor stability of chlorophyll, slow extraction speed, low measurement values, should be used with caution.

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