What are absorption, excitation and emission spectroscopy?

In the article, the difference between molecular absorption spectrum, excitation spectrum and emission spectrum is explained.

absorption spectrum

Figure 1: Absorption spectrum of anthracene in cyclohexane measured by fluorescence Spectrophotometer . Experimental parameters: Δλ = 1 nm.

Absorption spectroscopy (also known as UV-Vis spectroscopy, absorption spectroscopy, and electron spectroscopy) shows a sample's absorbance as a function of wavelength of incident light (Figure 1) and is measured using a Spectrophotometer . Absorption spectra are measured by varying the wavelength of the incident light using a monochromator and recording the intensity of the transmitted light on a Detector. The intensity of light sent through the sample, I sample, (eg analyte dissolved in solvent) and the intensity of light passing through a blank, I blank (solvent only) are recorded and the absorbance of the sample calculated using:

Absorbance is linearly proportional to the molar concentration of the sample. The concentration of the sample can then be calculated from the absorption spectrum using the Beer-Lambert law.

Figure 2: Schematic of measuring absorption spectra in a Spectrophotometer .

excitation spectrum

Figure 3: Fluorescence excitation spectrum of anthracene in cyclohexane measured by fluorescence Spectrophotometer . λem = 420 nm, Δλem = 1 nm, Δλex = 1 nm.

The fluorescence excitation spectrum shows the fluorescence intensity as a function of excitation light wavelength (Figure 3) and is measured using a spectrofluorometer. The wavelength of the emission monochromator is set to the known fluorescence emission wavelength of the sample, and the wavelength of the excitation monochromator is scanned over the desired excitation range, and the fluorescence intensity recorded on the Detector is recorded as a function of the excitation wavelength. If the sample follows Kasha's and Vavilov's laws, the excitation and absorption spectra will be the same (compare Figure 1 and Figure 3). The excitation spectrum can thus be considered as the absorption spectrum for fluorescence detection.

Figure 4: Schematic of measuring excitation spectra in a spectrofluorometer.

ll

Figure 5: Fluorescence emission spectra of anthracene in cyclohexane measured by a spectrofluorometer. λex = 340 nm, Δλex = 1nm, Δλem = 1 nm

Fluorescence emission spectra show the variation of fluorescence intensity with the wavelength of emitted light (Figure 5) and are measured using a spectrofluorometer. Set the wavelength of the excitation monochromator to the wavelength the sample is known to absorb and scan the wavelength of the emission monochromator over the desired emission range and record the fluorescence intensity at the Detector as a function of the emission wavelength.

Figure 6: Schematic for measuring emission spectra in a spectrofluorometer.