Excitation spectrum

An excitation spectrum records a sample's fluorescence signal as the excitation wavelength is varied while the observation band is held fixed. It asks which incident wavelengths efficiently produce the selected emission. An emission spectrum instead varies the observed emission wavelength for a fixed excitation setting.

A fluorometer implements the scan with its excitation monochromator while keeping the emission selection fixed. For example, observing near $520\mathrm{nm}$ while scanning excitation from $400$ to $490\mathrm{nm}$ defines an illustrative excitation scan; it is not a measurement of a named substance. Finite band widths and the instrument's wavelength-dependent response are part of the measurement.^[ref][ref]

For a homogeneous sample under a simple photon-balance model, writeHere $q$ is the incident photon rate, $A$ the absorbance, $\Phi$ the fluorescence quantum yield, and $K$ a constant detection fraction including the selected emission band. This model assumes that emitted light is not appreciably reabsorbed and that the collection and emission-band fractions remain constant.

When $A$ is small, $1-10^{-A}\simeq (\ln 10)A$. After correcting for the incident photon rate, the excitation shape then follows the absorption spectrum if $\Phi$ and the other stated factors stay constant. For $A=0.01$ and $0.02$ at two settings, the predicted corrected-signal ratio is approximately two; the exact absorbed-fraction ratio is about $1.98$. Unequal incident photon rates would change the raw ratio even for the same sample.

An excitation spectrum is consequently not automatically an absorption spectrum. Multiple emitters, wavelength-dependent yield and an inner filter effect can change the correspondence. JASCO's dilute-versus-concentrated comparison, monitored at $450\mathrm{nm}$ emission, illustrates concentration-dependent distortion of an excitation scan.^[ref]