Chapter 18

Spectrophotometry Fundamentals

  • Spectroscopy: theoretical approach to the science of studying the interaction between matter and radiated energy
  • Spectrometry: practical application of spectroscopy
    • uses spectrometer instruments
  • Spectrophotometry: method used to measure how much a chemical substand absorbs light as a beam of light passes through a sample solution
    • Spectrophotometers are instruments used to quantitaively measure the reflection or transmission properties of a material as a function of wavelength (a spectrum)
  • Spectrophotometric titrations: Change in absorbance is monitored during titration to tell when the equivalence point has been reached.

Properties of Light

  • Wave-particle duality of light
    • light behaves as a wave (with wavelength and frequency) and exhibits the wave phenomena of interference, diffraction, and reflection. Wave properties govern light behavior such as interference and diffraction
    • light behaves as a particle (a photon) that carries a discrete energy that can be absorbed or emitted by a molecule
  • Light waves consist of perpendicular, oscillating electric, and magnetic fields
    • wavelength (lambda): distance between wave crests
    • wavenumber: measure of spacial frequency
  • Speed of light
    • Frequency (v): number of oscillations per second of the wave
    • In a medium other than a vacuum, speed of light is c/n where n is the refractive index for light of wavelength traveling through the specified medium

Absorption of Light

  • Spectrophotometry: any technique that uses light to measure chemical concentrations
  • Absorption spectrophotometry: any technique that uses the absorption of light to measure chemical concentrations
  • Absorption of light by a molecule
    • absorbance is proportional to concentration of light-absorbing molecules
    • chromophore: part of molecule responsible for light absorption
    • molecular energy level diagrams describe the absorption of light by a molecule
    • molecule can only be in one of the energy states at a time
    • at ordinary temps, most molecules are found in the ground state
  • Absorption spectrometer
    • measures absorbance
    • components:
    • light source
      • lamp that produces light of many wavelengths
    • wavelength selector (monochromator)
      • breaks light into its colors, blocks all but a selected color
    • sample
      • P<Po when sample absorbing
      • P=Po when no light is lost in the sample
      • Po: irradiance of the monochromatic light as it enters the sample
      • P: irradiance of the monochromatic light as it exits the sample
    • light detector
      • measures irradiance of monochromatic light tranmitted through the sample
  • Key definition
    • Irradiance: energy per unit time per unit area in a light beam
    • Monochromatic light: consists of “one color” (one wavelength)
  • Absorption Spectrum and Color
    • any substance that absorbs visible light appears colored when white light is tranmitted through or reflected from it
    • Our eyes detect the wavelengths not absorbed
    • The obsorbed color is call the complement of the absorbed color

Measuring Absorbance

  • Cuvettes have flat, fused silica faces. Glass can be a cheaper alternative, but cannot be used to UV spectroscopy as glass absorbs UV radiation.
  • Handling of cuvettes
    • don’t touch clear face (fingerprints scatter and absorb light)
    • keep containers cover to exclude dust (dust scatters light)

What Happens When a Molecule Absorbs Light?

  • Single state: a state in which unpaired electrons have opposite spin
    • in a magnetic fied, it remains a single state
  • Triplet state: a state in which unpaired electrons have identical spin
    • in a magnetic field, it splits into three states
  • Infrared photons induce transitions between vibrational states of motion of a molecule
  • Microwave photons induce transitions between rotational states of a molecule
  • Internal conversion (IC): nonradiative transition between states will the same spin
  • Intersystem crossing (ISC): nonradiative transition between states with different spin
  • Fluroescence: release of photon by radiative relaxation (emitting a proton) from a singlet state to another singlet state
    • favored at lower temps
  • Phosphorescence: release of a photon by the radiative relaxation from a triplet state to a singlet state
    • favored at higher temps

Luminescence

  • Luminescence is the emission of light from an excited state molecule
  • Chemiluminescence is the emission of light from a chemical reaction
  • Rayleigh and Raman peaks show up in fluorescent spectra but are not due to fluorescence