UV-visible spectrophotometry

Explain how different wavelengths of light interact with matter, including the concept of resonance, different types of transitions, chromophores/auxochromes, and selection rules.

• resonance: energy matching
• quantum yield: fraction of excited states that do emit photon
• chromophore: light absorbing groups (part of molecule)
  • 200 - 800 nm: pi-electron and hetero atoms with non-bonding valence shell electron pairs
  • non bonding electrons in water, alcohols, either don’t absorb above 160nm, they are suitable solvents for spectroscopy
    • increasing/extending conjugation of unsaturated bonds decreases transition energies -> energy gap between HOMO and LUMO decreases
• auxochrome: chemical group attached to chromophore that modifies its light absorption (alter energy of MOs)
  • acid-base indicators: isobestic point -> total absorbance stays the same (?)
• non-boding electrons in O of water, alcohol, ether are good solvents for UV/Vis (don’t absorb above 160)

Nature of Shift	Term	How
longer wavelength	red shift	adding double bonds
shorter wavelength	blue shift
greater absorption	hyperchromic	doubles with each new conjugated double bond
lower absorption	hypochromic

• HOMO LUMO transitions:
  • in the UV/Vis range
  • singlet state:
    • ground: two electrons opposing spin, same orbital
    • excited: two electrons opposing spin, different orbitals

Draw and label a Jablonski energy level diagram and relate its features to absorption spectra.

• Bandwidths also depend on local environment (including effect of vib. & rotational levels)
  • we get absorption bands from electronic transitions, vibrational levels, rotational levels, other collections/interaction

Describe the processes that occur when UV-visible light is absorbed by a molecule, including excited states and relaxation processes.

• Relaxation pathways: fluorescence, internal conversion, intersystem crossing, phosphorescence
  • vibrational relaxation: energy lost to heat, electron stays in same electronic state
  • internal conversion: energy lost to heat, electron moves to lower electronic state, requires overlap between vibration levels and lower electronic state (horizontal energy transfer)
  • fluorescence: only occurs in singlet to singlet state
  • intersystem crossing: excited singlet move to excited triplet (or other way)
  • phosphorescence: only occurs in triplet to singlet state
  • light emitted from fluorescence or phosphorescence is always same or less than excitation wavelength
  • relative time frame:
    • absorption < vibration relaxation and internal conversion < fluorescence < phosphorescence (slow)

Explain the differences between atomic and molecular absorption spectra.

UV/Vis spectra for molecules/ions

• difference in energy between HOMO and LUMO -> UV/Vis, absorption of photon is possible
• types of transitions, n is non bonding:
  • σ → σ^*, 200 nm
  • n → σ^*, 160-260 nm
  • π → π^*, 200-500 nm
  • n → π^*, 250-600 nm
• charge transfer: inorganic metal-ligand complexes, electron from metal transferred to ligand -> produce very large absorbance
• UV/Vis more broad than IR
  • UV/Vis absorption results in change to electronic energy levels and maybe vibrational -> number of closely spaced absorption bands that merge together to form single broad absorption band
  • IR absorption only results in change to vibrational energy levels

UV/Vis spectra for atoms

• enough energy to cause change in atom’s valence electrons
• only allowed between $l +-1$$
• excited state lifetime is short
• narrow width in absorption lines, due to fixed difference in energy and lack of rotational/vibrational energy levels (width is 10⁻⁵ − 10⁻³ nm)

Explain the significance of the terms in the Beer-Lambert Law, and how they relate to molecular features and experimental design.

• light absorption: by chromophores, reflection, scattering loss
• molar absorption coefficient + path length => slope => sensitivity

Explain the limitations of Beer’s Law.

• real deviations:
  • high concentration: chromophores affect charge distribution of other chromophores
  • epsilon dependson refractive index
• apparent deviations:
  • light is not perfectly monochromatic

Utilize Beer’s Law for determining the concentration of one or more analyte species from one or more absorbance measurements.

• mixtures: A_total = A₁ + A₂ + ... = b(ϵ₁c₁ + ϵ₂c₂) + ...
• A_λ1 = b(ϵ_1λ1c₁ + ϵ_2λ1c₂) + ..., A_λ2 = b(ϵ_1λ2c₁ + ϵ_2λ2c₂) + ...
  • etc for the other wavelengths

Relate the choice and arrangement of components in a spectrophotomer to its capabilities, performance, advantages and disadvantages. Draw and label a detailed block diagram of a spectrophotometer, including the light path and components, and describe the function of each of those components.

single beam instrument

1. light source
   • D (UV)
   • T (Vis)
   • glass absorbs wavelength less than 350 nm, quartz absorbs below 190 nm
2. wavelength selector
   • diffraction grating, prism, filter
     • diffraction grating equation: a − b = mλ = d(sin(i) + sin(r))
       • where:
         • d: groove spacing (related to lines/mm)
         • i: incident angle
         • r: reflection angle
         • m: order (use filters to block unwanted)
   • monochromator: slit, diffraction grating, slit
     • narrow slit => S/N decreases, but bandwidth more narrow
     • double monochomator: reduce stray light + improve linearity => throughput decreased
       • does not remove unwanted orders (need filter)
       • slit => grating => slit => grating => slit
   • groove density increases (lines/mm) => dispersion increases => resolution increases => working range decreases (PDA detector)
3. Sample cell
4. Photodetector
   • PMT
   • Photodiode array
5. Computer

—

• UV/Vis diode array design
  1. dual lamp
  2. lens
  3. shutter
  4. sample
  5. lens
  6. slit
  7. grating
  8. PDA
• double beam: improve precision + S/N
  • sample and reference cell
  • compensate for flicker in lamp intensity

Describe the operation of key components for measuring absorbance:

Light sources (tungsten, deuterium)

• D (UV)
• T (Vis)

Monochromators (gratings and slits)

• filters have a fixed wavelength
• if we want to make measurements at different wavelengths -> need more than one filter
• monochromator: select narrow band of radiation, allow for continuous adjustment of band’s nominal wavelength
  • nominal wavelength: the wavelength you want?
    • want high throughput of radiation and narrow effective bandwidth

• collimating mirror: collects radiation
  • reflects parallel beam of radiation to diffraction grating
• differaction grating: optically reflecting surface with large number of parallel grooves
  • disperses radiation -> focused onto planar surface that contains exit slit
  • or prism
• converts polychromatic source of radiation to monochromatic source of finite bandwidth
• exit slit:
  • narrow: smaller effective bandwidth and better resolution, but smaller throughput of radiation
• can be fixed-wavelength or scanning
  • fixed: manually select wavelength by rotating grating

Sample cells (design and materials)

• glass (silicate): 400 - 2000
• glass (optical): 350 - 2000
• quartz: UV 200 - 3000
• plastic: vis only, 380 - 780

Photodetectors (PMT, photodiode, photodiode arrays)

• not equally sensitive to all wavelengths
  • measuring reference can help
• PMT
  • photoelectric effect: signal amplification with electron cascade
    • photocathode => focussing electrode => dynode(s) => anode
    • more dynodes => more sensitive
    • not equally sensitive to all wavelengths
• silicon photodiode
  • PN-junction in silicon
  • voltage is created
    • proportional to incident light intensity
• PDA (made of photodiode)
  • entire spectrum measured
  • PDA replaces exit slit => each PN element is a slit
  • don’t have great S/N

misc notes

textbook notes:

• if energy (ℏv) of photon is more than excited state - ground state, excitation occurs
• atom/molecule in excited state can emit photon of energy ℏv
• you don’t see the colors a substance absorbs

spectroscopy based on absorption

• absorbed wavelength intensities are attenuated
• for an analyte to absorb EMR:
  • there must be mechanism which EMR interacts with analyte -> UV/Vis changes energy of electrons, IR -> bond vibrational energy
  • photon energy must equal different in energy between two allowed energy states

1. IR spectra for molecules for polyatomic molecules:

   • energy for allowed vibration mode: $$E_v = v + \frac{1}{2} h v_o$$
     • fundamental: +/- 1
     • overtone: +/- 2,3

   1. transmittance and absorbance

      • transmittance: $$$T = \frac{P_T}{P_0}$

      

      • redefine P₀ from blank so we don’t need to care about loss of light from the source
      • absorbance is linear function of analyte concentration: $$A = -log T = - log \frac{P_T}{P_0}$$
        • require line source instead of continuum source because effective bandwidth is too large