My experience is with atomic spectroscopy of alkali atoms, I've recently been asked by a friend to help with some advice on analyzing molecular spectrophotometry data in the context of molecular biology,
To fit spectroscopic data to extract density in atomic spectroscopy you typically need to know the relative transition strengths and natural linewidths for all transitions (for hyperfine resolution spectroscopy), know the interaction path length, know the doppler broadening coefficient (and know the temperature), construct a voigt profile and fit.
If I wanted to fit spectroscopic data to extract, say, ATP concentration from a spectrophotometer sweep, would I be able to do this by determining the corresponding molecular parameters? Are there caveats to single transition molecular spectroscopy that I should be aware of? It would seem that doppler broadening would be the largest contribution to the linewidth so it I may be able to fit to a simple gaussian profile instead of the more computational intense voigt.
In atomic spectroscopy you usually measure a change in coherent beam intensity so the contribution from fluorescence of the atom can be made negligible in the low power unsaturated regime with polarization analyzers and other small trade-tricks. I'm not completely certain how spectrophotometers are set up with that regards, would fluorescence near the measurement frequency be something I need to take into account?