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 108 m/s.
oIn a medium other than a vacuum, the speed of light is c/n, where n is the
oFor 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 10-34 Js.
oE = hc/v = hcṽ, where ṽ is 1/ λ or the wavenumber.
oEnergy is inversely proportional to wavelength and directly proportional to
oElectromagnetic 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
•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.
oA = log(Po/P) = -logT
oBeer’s Law: A = Ԑbc, where Ԑ is the molar absorptivity and has units of M-1cm-
oThe greater the molar absorptivity, the greater the absorbance.
oAbsorption Spectrum: a graph showing how A or Ԑ varies with wavelength.
oChromophore: 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
Spectrophotometry: any technique that uses light to measure chemical concentrations: colorimetry: a procedure based on absorption of visible light, wavelength ( ): the crest-to-crest distance between waves. 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. Irradiance/intensity/radiant (p): the energy per unit time per unit area in the light beam (w/m2): monochromator: a wavelength selector in which light passes through and it selects one wavelength. Transmittance (t): the fraction of original light that passes through the sample (t = P/po) and has a range of 0 to 1. 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.