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Topic 7.1
Principles of Spectrophotometry
Much of what we know about the photosynthetic apparatus was learned through spectroscopy—
that is, measurements of the interaction of light and molecules. Spectrophotometry is an
important branch of spectroscopy that focuses on the technique of measurement. Here we will
examine four topics: Beer's law, the measurement of absorbance, action spectra, and difference
spectra.

Beer's Law
An essential piece of information about any molecular species is how much of it is present.
Quantitative measures of concentration are one of the cornerstones of biological science. Of all
the methods that have been devised for measuring concentration, by far the most widely applied
is absorption spectrophotometry. In this technique, the amount of light that a sample absorbs at a
particular wavelength is measured and used to determine the concentration of the sample by
comparison with appropriate standards or reference data. The most useful measure of light
absorption is the absorbance (A), also commonly called the optical density (OD) (Web Figure
7.1.A). The absorbance is defined as A = log I0 / I where I0 is the intensity of light that is incident
on the sample and I is the intensity of light that is transmitted by the sample.

Web Figure 7.1.A Definition of absorbance. A monochromatic incident light beam of intensity
I0 traverses a sample contained in a cuvette of length (l). Some of the light is absorbed by the
chromophores in the sample, and the intensity of light that emerges is I.
The absorbance of a sample can be related to the concentration of the absorbing species through
Beer's law:

A = ε cl

where c is concentration, usually measured in moles per liter; l is the length of the light path,
usually 1 cm; and ε is a proportionality constant known as the molar extinction coefficient, with
the units of liters per mole per centimeter. The value of ε is a function of both the particular
compound being measured and the wavelength. Chlorophylls typically have an ε value of about
100,000 L mol–1 cm–1. When more than one component of a complex mixture absorbs at a given
wavelength, the absorbances due to the individual components are generally additive.

The Spectrophotometer
The absorbance is measured by an instrument called a spectrophotometer (Web Figure 7.1.B).
The essential parts of a spectrophotometer include a light source, a wavelength selection device
such as a monochromator or filter, a sample chamber, a light detector, and a readout device,
usually also include a computer, which is used for storage and analysis of the spectra. The most
useful machines scan the wavelength of the light that is incident on the sample and produce, as
output, spectra of absorbance versus wavelength, such as those shown in textbook Figure 7.7.

Web Figure 7.1.B Schematic diagram of a spectrophotometer. The instrument consists of a

light source, a monochromator that contains a wavelength selection device such as a prism, a
sample holder, a photodetector, and a recorder or computer. The output wavelength of the
monochromator can be changed by rotation of the prism; the graph of absorbance versus
wavelength is called a spectrum.

Action Spectra
The use of action spectra has been central to the development of our current understanding of
photosynthesis. An action spectrum is a graph of the magnitude of the biological effect observed
as a function of wavelength. Examples of effects measured by action spectra are oxygen
evolution (Web Figure 7.1.C) and hormonal growth responses due to the action of phytochrome
(see Chapter 16 of the textbook). Often an action spectrum can identify the chromophore
responsible for a particular light-induced phenomenon. Action spectra were instrumental in the
discovery of the existence of the two photosystems in O2-evolving photosynthetic organisms.
Web Figure 7.1.C An action spectrum compared to an absorption spectrum. The absorption
spectrum is measured as shown in Web Figure 7.1.B. An action spectrum is measured by
plotting a response to light such as oxygen evolution, as a function of wavelength. If the
pigments used to obtain the absorption spectrum are the same as those that cause the response,
the absorption and action spectra will match. In the example shown here, the action spectrum for
oxygen evolution matches the absorption spectrum of intact chloroplasts quite well, indicating
that light absorption by the chlorophylls mediates oxygen evolution. Discrepancies are found in
the region of carotenoid absorption, from 450 to 550 nm, indicating that energy transfer from
carotenoids to chlorophylls is not as effective as energy transfer between chlorophylls.

Some of the first action spectra were measured by T. W. Engelmann in the late 1800s (Web
Figure 7.1.D). Engelmann used a prism to disperse sunlight into a rainbow that was allowed to
fall on an aquatic algal filament. A population of O2-seeking bacteria was introduced into the
system. The bacteria congregated in the regions of the filaments that evolved the most O2. These
were the regions illuminated by blue light and red light, which are strongly absorbed by
chlorophyll. Today, action spectra can be measured in room-sized spectrographs in which the
scientist enters a huge monochromator and places samples for irradiation in a large area of the
room bathed by monochromatic light. But the principle of the experiment is the same as that of
Engelmann's experiments.
Web Figure 7.1.D Schematic diagram of the action spectrum measurements by T. W.
Engelmann. Engelmann projected a spectrum of light onto the spiral chloroplast of the
filamentous green alga Spirogyra and observed that oxygen-seeking bacteria introduced into the
system collected in the region of the spectrum where chlorophyll pigments absorb. This action
spectrum gave the first indication of the effectiveness of light absorbed by accessory pigments in
driving photosynthesis.

Difference Spectra
An important technique in studies of photosynthesis is light-induced difference spectroscopy,
which measures changes in absorbance (Web Figure 7.1.E). In this technique, bright light, often
called actinic light, is used to illuminate a sample, while a dim beam of light is used to measure
the absorbance of the sample at wavelengths other than that of the actinic beam. In this way a
difference spectrum is obtained, which represents the changes in the absorption spectrum of the
sample induced by illumination with the actinic light. Absorption bands that disappear upon
illumination appear as negative peaks; new bands that appear upon illumination appear as
positive peaks. Difference spectra give important clues to the identity of molecular species
participating in the photoreactions of photosynthesis. The difference spectrum of the
photooxidation of P700 (a chlorophyll that absorbs light of wavelength 700 nm, see textbook
Figure 7.19) is shown in Web Figure 7.1.F (Ke 1973).

Web Figure 7.1.E Principle of difference spectroscopy. We measure difference spectra by

observing as a function of time the change in the absorbance of a measuring light λ1 in a sample
when an actinic light is turned on. The actinic light λ2 causes chemical changes in the sample that
change its absorption spectrum. Blocking filters are necessary to prevent scattered actinic light
from entering the detector. The up and down arrows signify the times when the actinic light was
turned on and off, respectively. The change in absorbance induced by the actinic light, ΔA, can
be either positive or negative. Usually, measurements are made at one wavelength at a time. A
difference spectrum is built up by repetition of the measurement at many different wavelengths.

Web Figure 7.1.F Light-minus-dark difference spectrum for photooxidation of P700, measured
as shown in Web Figure 7.1.E. The decreases of absorption (bleaching) at 430 and 700 nm are
due to loss of the absorbance of P700. Increases observed around 450 nm and beyond 730 nm are
due to absorption by P700+ (oxidized P700). (After Ke 1973.)

By the use of special flash techniques, it is possible to record the difference spectrum at a given
time after flash excitation. Multiple difference spectra recorded at different times after flash
excitation can be used to measure the kinetics of the chemical reactions that follow photon
excitation of a reaction center. These techniques can have extraordinary time resolution, in some
cases less than a picosecond (10–12 s), and have provided great insights into the earliest events in
the photosynthetic energy storage process.

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