5
I
NTRODUCTION
Theory
A spectrophotometer is primarily used to identify
unknown substances and determine their con-
centrations. The following principles outline how
this is accomplished.
Matter absorbs energy when it interacts with an
energy source such as light. Due to its distinctive
atomic structure, each substance only absorbs
energy between certain levels. Since energy is
inversely proportional to wavelength, E=hc/
λ
(where h is Plank’s constant, c is the speed of
light, and
λ
is the wavelength of light), every
substance has a characteristic absorption spec-
trum.
For example, hydrogen absorbs light at the fol-
lowing wavelengths in the visible region:
410.7nm, 433.8nm, 485.7nm and 657.9nm. We
can verify the existence of hydrogen in an un-
known sample by comparing the wavelengths
actually absorbed by the unknown sample to
hydrogen’s known absorption spectrum.
Therefore, a device, namely your spectrophotom-
eter, is required to measure absorption spec-
trums in order to identify an unknown substance.
A spectrophotometer measures the amount of
light absorbed at distinct wavelengths of incident
light. As a result, a graph of the amount of ab-
sorbed light at various wavelengths can be plot-
ted (absorbance vs. wavelength, as shown in Fig.
1.) This is known as an absorption spectrum.
Such a graph can be used to identify the pres-
ence of a particular substance.
The concentration of an unknown sample can
also be determined. When a light beam is inci-
dent to a sample, part is absorbed and part is
Fig. 1
