Chlorophyll - Spectrophotometry

Spectrophotometry

Measurement of the absorption of light is complicated by the solvent used to extract it from plant material, which affects the values obtained,

In diethyl ether, chlorophyll a has approximate absorbance maxima of 430 nm and 662 nm, while chlorophyll b has approximate maxima of 453 nm and 642 nm.
The absorption peaks of chlorophyll a are at 665 nm and 465 nm. Chlorophyll a fluoresces at 673 nm (maximum) and 726 nm. The peak molar absorption coefficient of chlorophyll a exceeds 105 M−1 cm−1, which is among the highest for small-molecule organic compounds.
In 90% acetone-water, the peak absorption wavelengths of chlorophyll a are 430 nm and 664 nm; peaks for chlorophyll b are 460 nm and 647 nm; peaks for chlorophyll c1 are 442 nm and 630 nm; peaks for chlorophyll c2 are 444 nm and 630 nm; peaks for chlorophyll d are 401 nm, 455 nm and 696 nm.

By measuring the absorption of light in the red and far red regions it is possible to estimate the concentration of chlorophyll within a leaf.

While absorption instruments have been proven over time to work well as a non-destructive test on many types of samples, they do have limitations. Absorption technique limitations:

The sample to be measured must completely cover the instrument’s aperture, with no holes.
Samples must be thin enough to allow transmission of the measuring wavelengths of light.
Surfaces must be relatively flat and uniform.
Variable fluorescence (the Kautsky induction effect) caused by the red wavelength of absorption instruments, limits the repeatability of measurements at the same location.
Selection of the measuring area, on smaller leaves, can cause measurement variations due to internal leaf structure variation. Veins and midribs can cause significant variation on samples.
Wavelengths used in absorption methods, typically limit linear correlation with chemical chlorophyll content methods to concentration levels below 300 mg/m2.

As a result, absorption instruments do not work with conifer needles, turf grasses, Arabidopsis leaves, moss, most CAM plants such as prickly pear cactus, and Agave, fruit, stems, petioles, lichens, and algae on rocks. Furthermore, it is difficult to get reliable readings on very small leaf plants such as immature rice and wheat.

A superior method is to measure the chlorophyll content using chlorophyll fluorescence. Gitelson (1999) states, "The ratio between chlorophyll fluorescence, at 735 nm and the wavelength range 700nm to 710 nm, F735/F700 was found to be linearly proportional to the chlorophyll content (with determination coefficient, r2, more than 0.95) and thus this ratio can be used as a precise indicator of chlorophyll content in plant leaves." Non-destructive, hand-held meters use this effect to measure the chlorophyll content of samples where traditional absorbance techniques cannot be used. This new method has many advantages over older, absorption techniques. These include:-

Samples do not need to fill the measuring aperture, so small samples can be measured.
Samples do not need to allow transmission of the measuring beam, so thick samples can be measured.
Surfaces do not need to be flat and uniform, so difficult plant morphologies can be measured.
No Kautsky induction effect, so measurements can be repeated rapidly at the same sight.
Small sample area, so naturally occurring structures like mid ribs and veins can be avoided.
Higher measuring range (675 mg/m2, as opposed to 300 mg/m2 with absorption techniques), so many more samples can be measured.

By measuring chlorophyll fluorescence, plant ecophysiology can be investigated. Chlorophyll fluorometers are used by plant researchers to assess plant stress.