chm317 spectroscopy unit

spectroscopy

• is based on light-matter interactions

• energy + intensity of light absorbed, emitted, or scattered —> shows the set of allowed states —> helps identify matter + amount

absorbance spectroscopy

• useful for both atomic + molec identification (using spectra)

• and for quantitation (using Beer’s law) bc matter gains energy due to electronic and/or vib. absorptions - amount of light absorbed proportional to the concentration

how can absorbance spec be classified + what happens in UV-vis/elec spec vs. IR/vib spec

• absorbance spec can be classified by range of EM spectrum used or type of resonant transitions occurring

• UV-vis/electronic spectroscopy uses UV/vis radiation to resonantly excite electronic transitions, changing electron occupancy in molecular orbitals and therefore corresponding electronic states

  • eg, when Na absorbs photons, there is a 3s to 3p transition, and electronic state changes from D0 to D1

  • eg, Rhodamine B absorbs photons, HOMO to LUMO transition, electronic state changes from S0 to S1

• IR/vib. spectroscopy uses lower E light to resonantly excite vibrational and rotational (nuclear) motions in molecules

UV/vis absorbance transitions

• recall: UV/vis transitions involve change in electron occupancy in molecular orbitals and therefore electronic state changes (electronic excitation)

• transitions may also include change in vibrational levels (vibronic excitation)

• molar absorptivity ε(λ) is a measure of how strongly a compound absorbs light at a given wavelength —> this is proportional to the probability of transition (how likely electronic transition occurs)

IR absorbance transitions

• vibronic transitions are lower in E than electronic transitions

• energy changes in matter occur due to vib transitions in the IR region

• eg, bond stretching, bending, scissoring, etc.

transmittance in absorbance spec

• we track fraction of power lost as light passes through a sample

• transmittance (T) = P/P0

  • power through sample cell over power through blank cell (containing only matrix) to account for phenomena that causes loss of power (like scattering)

• data often reported in absorbance (A)

  • A = -logT = -log(P/P0) = log(P0/P)

relationship between transmittance and absorbance

• high T means most of the light passes through, which means low A

• low T (P/P0) means little light passes through, which means high A

Beer-Lambert law - relationship between A, T, and concentration

• A = log(P0/P) = εbc (molar absorptivity (L mol-1 cm-1) x path length of cuvette (cm) x concentration (M)

• T = P/P0 = 10^-A = 10^-εbc

• if sample has a well-defined path length, there is linear dependence of absorbance on concentration at a given wavelength, and we can quantify concentration (there are limitations though)

absorbance spectra

• spectra shows probability matter will have lower E state to higher E state transitions upon photon absorption

• good chromophores usually have ε(max) of 10^4 to 10^5 L mol-1 cm-1 at electronic/UV/vis absorbance maxima

• spectral features / bands = possibly correspond to excitation from ground elec state to several different excited elec states (elec transitions) or to diff vib levels within an excited elec state (vibronic transitions)

limitations of absorbance spec for quantitation

• range of linearity is limited (absorbance measurements work best between 10^-4 to 2.0

• Caveat 1: Beer-Lambert law is strictly true only for an infinitely narrow wavelength, but wavelengths selectors use a small range of wavelengths Δλeff, so if A isn’t constant over the bandwidth, the B-L relationship is non-linear

  • best to choose the narrowest bandwidth at wavelength of maximum A (λmax) that is ~constant with respect to λ

• Caveat 2: interferences - if there are multiple absorbing species present, total absorbance is the sum of each species’ absorbance A_λ​ = ε₁​bc₁​ + ε₂​bc₂​ +… —> this is fine for atomic absorbance which have narrow peaks, but for molecular absorbance, there are many overlapping broad peaks (especially in higher E regions) that can affect quantitation

  • best to use separation methods (like HPLC before taking UV-Vis spectra) - usually done by selecting a wavelength common to analytes of interest so they can all be detected, but taking spectra over range of λ obtains more information

absorbance spec for trace analysis

• not a good choice for trace analysis (< nM or ppb)

• P0 and P may both be large which fine, but absorbance depends on the small diff b/w 2 large signals and noise may make small absorbance differences hard to measure, especially with small sample volumes that limits path length