Week 5 - Spectrophotometry summary

The Atom: Quantum Theory

  • Electrons and Orbits

    • Electrons orbit nucleus at specific sizes and energy levels.

    • Energy related to orbit size; radiation absorbed/emitted when electron moves or 'jumps' between orbits.

  • Bohr Model

    • Electrons in "allowed" orbits do not radiate energy.

    • Specific orbits correspond to defined energy states (quantum states); intermediate orbits do not exist.

  • Transitions and Energy Levels

    • Electron transitions between orbits result in emission or absorption of energy (e.g., Balmer series in hydrogen).

    • Photon energy: E = h
      u, where (h) is Planck's constant and (\nu) is frequency.

  • Photons and Electromagnetic Radiation

    • Light (visible, ultraviolet, infrared) behaves as both a particle (photon) and a wave.

    • Photon is the fundamental unit of electromagnetic radiation, quantified in moles (e.g., 1 mol = 6.02 \times 10^{23} photons).

  • Electromagnetic Spectrum

    • Ranges from radio waves (long wavelength, low energy) to gamma rays (short wavelength, high energy).

    • Visible light spans 400 - 800 nm, with colours associated with specific wavelengths.

  • Spectrophotometry Principles

    • Involves measuring absorbance and transmission properties of a solution.

    • Uses Lambert's and Beer's laws to relate absorbance to concentration and path length.

  • Beer-Lambert Law

    • A = -\log_{10}(I_1/I_0) = \varepsilon bc, where (A) is absorbance, (I_0) is incident light intensity, (I_1) is transmitted light intensity, (\varepsilon) is molar extinction coefficient, and (b) is path length.

    • Characteristic absorption spectra for each substance enable identification and quantification.

  • Applications of Spectrophotometry

    • Used in qualitative analysis (identifying compounds) and quantifying nucleic acids (e.g., DNA) based on absorbance at 260 nm.

    • Contaminants can affect measurements; purity assessed via OD260/OD280 ratio (pure DNA ~1.8, RNA ~2.0).

    • Extended applications include ELISA, FTIR for material analysis, and flow cytometry for cell analysis.