Chapter 17: Fundamentals of Spectrophotometry
Properties of Light
• Spectrophotometry: any technique that uses light to measure chemical
• Colorimetry: a procedure based on absorption of visible light.
• Wavelength (λ): the crest-to-crest distance between waves.
• Frequency (v): the number of complete oscillations that the wave makes each
• Relation between frequency and wavelength: λv = c, where c is the speed of light at
2.998 x 10 m/s.
o In a medium other than a vacuum, the speed of light is c/n, where n is the
o For visible wavelengths in most substances, n > 1, so visible light travels
more slowly through matter than through vacuum.
• Photons: particles of light with regard to energy.
• Relation between energy and frequency: E = hv, where h is Planck’s constant of
6.626 x 10 -34Js.
o E = hc/v = hcṽ, where ṽ is 1/ λ or the wavenumber.
o Energy is inversely proportional to wavelength and directly proportional to
o Electromagnetic Spectrum: the visible spectrum spans the wavelength range
of 380-780 nm.
Absorption of Light
• Excited State: when a molecule absorbs a photon, the energy of the molecule
increases and the molecule is promoted to an excited state.
• Ground State: if a molecule emits a photon, the energy of the molecule is lowered
and this is the lowest energy state of a molecule.
• Irradiance/Intensity/Radiant (P): the energy per unit time per unit area in the light
beam (W/m ).
• Monochromator: a wavelength selector in which light passes through and it selects
• Transmittance (T): the fraction of original light that passes through the sample (T =
P/Po) and has a range of 0 to 1.
• Percent Transmittance: 100T and ranges between 0 to 100%.
• Absorbance: directly proportional to the concentration of the light-absorbing species
in the sample.
o A = log(P oP) = -logT
o Beer’s Law: A = Ԑbc, where Ԑ is the molar absorptivity and has units of M cm
o The greater the molar absorptivity, the greater the absorbance.
o Absorption Spectrum: a graph showing how A or Ԑ varies with wavelength.
o Chromophore: the part of a molecule responsible for light absorption.
• Any substance that absorbs visible light appears colored when white light is
transmitted through it or reflected from it; the substance absorbs certain wavelengths of the white light and our eyes detect the wavelengths that are not
• When Beer’s Law Fails
o At very high concentration, the solute becomes the solvent so the properties
of a molecule are not exactly the same in different solvents.
o Nonabsorbing solutes in a solution can also interact with the absorbing
species and alter the absorptivity.
o Beer’s Law works for monochromatic radiation passing through a dilute
solution in which the absorbing species is not participating in a concentration-
• Cuvet: where liquid samples are usually contained and has flat, fused-silica faces.
• Do not touch the clear faces of a cuvet because fingerprints scatter and absorb light.
• Gases are more dilute than liquids and require cells with longer pathlengths (10cm to
• For spectrophotometric analysis, we choose the wavelength of maximum absorbance
o The sensitivity of the analysis is greatest at maximum absorbance; we get the
maximum response for a given concentration of analyte.
o The curve is relatively flat at the maximum, so there is little variation in
absorbance if the monochromator drifts a little or if the width of the
transmitted band changes slightly.
• Modern spectrophotometers are most precise at intermediate levels of absorbance,
around 0.3 to 2.
Beer’s Law in Chemical Analysis
• Proteins are normally assayed in the ultraviolet region at 280 nm because aromatic
groups present in virtually every protein have an absorbance maximum at 280 nm.
• Reagent Blank: contains all reagents but with analyte replaced by distilled water.
• Subtract the blank absorbance from the absorbance of samples and standards before
doing any calculations.
• Supernate: the liquid layer above the solid that collects at the bottom of a tube
• Spectrophotometric Titration: we monitor changes in absorbance during a titration to
tell when the equivalence point has