13
4. Once the optimum position has been obtained, verify that the lamp chamber
door is secure and all items are returned to their original state except the lamp
bracket position.
Absorbance Accuracy and Stray Light Checking
Specification: ± 2% at 1A (100DPlus), ± 1% at 1A and 2A.
The absorbance accuracy and Stray Light should be checked against a set of
neutral density filters accurately calibrated to the NIST standards. Contact
Customer Service for more information.
Sample Experiments
Experiment 1
A Brief Introduction to Beer’s Law
A spectrophotometer is primarily used to identify unknown substances and to
determine their concentrations. The following principles outline how this is
accomplished.
Matter absorbs energy when it interacts with an energy source such as sound or
light. Due to its distinctive atomic structure, each substance only absorbs energy at
specific frequencies.
Since energy is proportional to frequency, E=hc/λ (where
h=6.6262 x 10
–34
Joules =
Plank’s constant, c=2.998 x 10
8
m/s = the speed of light
in a vacuum (often rounded to 3.00 x 10
8
m/s), and λ=wavelength of light). Every
substance has a characteristic absorbance of the light spectrum (which means that
there are wavelengths where the atoms of a substance become highly excited).
For example, hydrogen (H
2
) absorbs light at the following wavelengths in the visible
region: 410.7 nm, 433.8 nm, 485.7 nm, and 657.9 nm. We can verify the existence
of hydrogen (H
2
) in an unknown sample by comparing the wavelengths absorbed by
the unknown sample to the wavelengths at which hydrogen absorption is known.
A spectrophotometer is required to measure absorption spectrums in order to
identify an unknown substance. The spectrophotometer is used to measure the
amount of light absorbed at distinct wavelengths. This can be plotted and the graph
can be used to identify the presence of a particular substance.
A spectrophotometer can also be used to determine the concentration of an
unknown sample. When a light beam is incident to a sample, part is absorbed, and
part is transmitted. The transmittance (T) is defined as the ratio of the transmitted
intensity of the light beam (X) to the initial intensity of the light beam (Y), or:
T=X/Y.
