Agriculture Reference
In-Depth Information
organic matter may have dark-colored components that are released into the
extracting solution and cause interference with the spectrophotometric
analysis. Thus extracts must be analyzed to make sure that they are free of
interference.
8.8.
COLOR MEASUREMENT: THE SPECTROPHOTOMETER
Color measurement is based on the Beer-Lambert law, which can be
expressed as follows:
I
A
=
log
10
I
ex
In this equation
A
is the absorbence also called the optical density,
I
is the
intensity of the bean of light entering the sample, and
I
ex
is the intensity of the
beam of light exiting the sample. The absorbance is proportional to both
the concentration of absorbing species and the pathlength of the light through
the sample. Normally standard-size sample cells are used, and so the path-
length is constant for all samples.
This type of analysis involves colored solutions; thus the spectrophotometer
need function only in the visible range of the spectrum. A visible light source
is used to provide light that passes through the sample in a cuvette, to a grating
where it is dispersed into its respective wavelengths. The analytical wave-
length, that is, the wavelength absorbed by the colored compound, is isolated
and directed to the detector, where the amount of light at this wavelength is
measured and displayed on the readout. A diagram of a spectrometer is shown
in Figure 8.8. Note that there are many different ways to design a spec-
trophotometer and its light path. The diagram given is simply a general rep-
resentation of how a spectrophotometer works.
Cuvettes used with the spectrophotometer are not simply test tubes. They
are specially made tubes and are often matched such that a set of tubes will
all have the same absorbence characteristics. Cuvettes should never be used
as test tubes; they must be kept clean at all times, and care must be taken not
Read out
0.55
Detector
Sample
cuvette
Slit
Grating
Light source
Figure 8.8.
Diagram of the light path in a spectrophotometer.
