Introduction to
Spectrophotometry & Beer’s Law
AP Chemistry
Mrs. Weston
What is spectrophotometry?
• A type of spectroscopy that studies the
transmission or reflection of different
frequencies of the electromagnetic spectrum
(specifically visible, near-IR and near-UV
ranges) by a sample of matter (usually a soln)
Other types of spectroscopy
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Atomic absorption spectroscopy
Atomic emission spectroscopy
Mass spectroscopy
Nuclear magnetic resonance (NMR)
Fourier transform infrared (FTIR) spectroscopy
Spectrophotometer
• Measures absorbance or
transmittance of light as a
function of wavelength
• Sample is placed into cuvette
• Light of selected wavelength is
passed through sample
• Amount of light absorbed is
measured
• Because other compounds in a solution (or
the solvent itself) may absorb the same
wavelengths as the compound being analyzed,
the absorbance of the sample is compared to
a reference blank.
• Ideally, the reference blank should contain
everything found in the sample solution except
the substance being analyzed.
• When using a Spec 20, every time the
wavelength is changed, the instrument needs
to be re-zeroed.
How it works
Transmission = I/Io
Absorbance = log T
How to choose wavelength
• We want to analyze samples using the
wavelength at which the most light is
absorbed – Wavelength of maximum
absorption (λmax)
Beer’s Law
• Amount of light absorbed is proportional to
the concentration of the solution
A = abc
• A = absorbance
• a = proportionality constant
(ε = molar absorptivity)
• b = path length (same for entire experiment)
• c = concentration (M)
What does this tell us?
• There is a direct relationship between
absorbance and concentration.
• When we prepare solutions of known
concentration and analyze them at λmax, we
can plot absorbance as a function of
concentration.
• The concentration of the unknown can be
determined by finding its absorbance and
plugging it into the equation for the best fit
line.
y = mx + b
Uses of spectrophotometry
• Determining concentration of any colored
solution (Fe, Cu, Co, Ni, MnO4, etc)
• Biology
• Biochemistry
• Forensic science