= 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.<Principles of fluorescence spectroscopy (3) - Instrument configuration by JASCO>[Optical system and Fig. 7, separate excitation and emission wavelength selection]{ref}<Principles of fluorescence spectroscopy (4) - Notes on fluorescence measurements by JASCO>[Spectral correction, instrument response versus sample spectrum]{ref}
For a homogeneous sample under a simple photon-balance model, write
$$
S(\lambda)=K\,q(\lambda)\,\Phi(\lambda)\bigl(1-10^{-A(\lambda)}\bigr).
$$
Here $q$ 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, $1-10^{-A}\simeq(\ln10)A$. 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.<Principles of fluorescence spectroscopy (4) - Notes on fluorescence measurements by JASCO>[Internal shielding effect and Fig. 10, dilute and concentrated sample excitation spectra at fixed emission]{ref}
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