Shines a beam of light on a sample.
The molecules in the sample interact
with the light waves in of 3 ways:
Absorb the energy
Reflect the energy
Transmit the energy between and
through the atoms and molecules of
Blue molecules absorb the other
colors of visible light.
Blue molecules are blue because
they reflect blue light.
Consider blue molecules, all the
wavelengths of light are absorbed,
except for the blue ones.
The blue wavelengths are
transmitted or reflected off the
molecules. If these blue
wavelengths hit a detector (such as
in the spectrophotometer or the
nerve cells in your eye), they appear
Molecules are whatever color of
light that they do not absorb.
Green molecules appear green
because they absorb most
wavelengths of visible light,
except the green wavelengths.
The spectrophotometer measures
the amount of light transmitted
through the sample
By using an equation (Beers law),
it converts the transmittance
data to an absorbance value.
What kind of data is this?
The concentration of an unknown
sample can be determined by
comparing the absorbance data to
standards of known concentration.
The data generated with the set of
known standards is called a
Prism or grating that direct
light of a specific wavelength.
Contains a tungsten lamp that
produces wwhhiittee lliightght.
Contains a deuterium lamp
that produces light in the UV
light part of the spectrum.
Knobs or buttons used to calibrate the
spec to measure the designated molecule.
White light hits the prism or
grating, it is split into the
colors of the rainbow (Visible
The wavelength knob rotates
the prism/grating, directing
different color of light toward
The wavelength of light
produced by the tungsten lamp
range from about 350 nm (Violet
light) to 700 nm (red light).
The molecules in the sample
either absorb or Transmit the
light energy of one wavelength
The detector measures the
amount of light being
transmitted by the sample and
reports that value directly (%
transmittance) or converts it to
the amount of light absorbed in
absorbance units (au) using
After collecting data for your
concentration an absorption
spectrum graph is created.
These can be used when
attempting to identify unknown
substances (e.g. CSI)
The absorbance spectrum is a
graph of a sample’s absorbance
at different wavelengths.
The spectrophotometer can
measure the amount of absorbance
lack of absorbance of different
colored light for a given
The concentration of molecules
in a solution affects the
Remember [ ] is a ratio – when we
change one number – it affects the
If there are more molecules in one
solution than in another, than there
are more molecules to absorb the
Determines the presence and
concentrations of samples.
Determines the purity of a
Look at the change of
samples over time.
A. Measure the absorbance of standards
containing known concentrations of the
B. Plot a standard curve with absorbance
on the X axis and analyte concentration on
the Y axis
C. Measure the absorbance of the
D. Determine the concentration of material
of interest in the unknowns based on the
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