A molecule irradiated with a beam of light in the ultraviolet and visible (UV-Vis) range might be temporarily raised from the lowest energy state (ground state) to a higher energy state (excited state). For the molecule to undergo an electronic transition, the amount of energy of the incident photons must be equal to the energy difference between the two electronic states involved (ground state and excited state), see Figure 1.

When a beam of monochromatic radiation (the incident beam intensity) passes through a homogeneous absorbing sample, the intensity of the light emerging from the sample (the transmitted intensity) can be reduced, with the transmitted intensity being lower than the incident intensity. If no radiation is absorbed by the sample and neglecting reflection losses, the incident intensity is equal to the transmitted intensity.

Figure 1 – Molecular absorption process between the ground and excited state.

The quantity that can be measured in a spectrometer, when studying the absorption of radiant energy by a sample, is called absorbance, A, and can be defined on the basis of the Beer-Lambert law (also known as Beer’s law) that is given by the following equation

where I_o and I_T are the intensities of the incident and transmitted radiation, respectively, ε (M^-1 cm^-1) is the molar absorption coefficient (absorptivity) of the molecule, l (cm) is the optical path of the solution (cuvette length) and (M) is the molar concentration of the absorption species in solution, see Figure 2.

The Beer-Lambert law shows mathematically that there is a linear relationship between absorbance and the molar concentration of the absorbing species if the path length and the wavelength of the incident radiation are kept constant.

The Beer’s law form the basis for quantitative study of the concentration of an absorbing specie in solution by measuring the amount of absorbed radiation. Despite being mathematically a linear relation, deviations from linearity are common when high concentrations or scattering species are present, which results in a nonlinear response. Another source of deviations from the Beer’s law may also be expected when the amount of stray light inside the spectrometer is high.

Figure 2 – Absorption of radiant energy that goes through a transparent sample, with a finite path length.

The quantitative absorption of radiant energy by a absorbing specie can also be defined using another quantity, the transmittance, T, which can be related to A through the following equation

The transmittance is the fraction of the incident light that passes through the sample. Therefore, the range of values for T is from 0 (for a totally absorbent solution) to 1 (for a totally transparent solution). It is also common to represent the transmittance values in a percentage scale, which equals T×100.

The absorption spectrum of a sample is normally represented by plotting the absorbance as a function of wavelength; additionally, is also common to represent the absorbing properties of a sample by plotting the molar absorption coefficient as a function of wavelength. The molar absorption coefficient measures the ability of the absorbing species in the sample to absorb light at a particular wavelength; a high molar absorption coefficient reflects a high probability of the transition to occur by plotting the molar absorption coefficient as a function of wavelength, see Figure 3.

Figure 3 – Typical Plot of an Absorption Spectrum.

The absorption and transmittance setup using cuvettes combines a DW light source (LS-DW) with a standard cuvette holder (SCH), set into a pass-thought configuration (a solid support holder can be used for solid transmittance measurements), and a FLEX spectrometer (UV-Vis-NIR configured). Find out more about our pre-configured packages for UV-Vis-NIR absorbance & transmittance measurements.

The absorption and transmittance setup using flow cells combines a DW light source, LS-DW, with an absorbance flow cell, set into a Z-shaped configuration, and a FLEX spectrometer (UV-Vis-NIR configured). Find out more about our pre-configured packages for UV-Vis-NIR absorbance and transmittance measurements.

The absorption and transmittance setup using a absorbance/transmittance probe combines a DW light source, LS-DW, with an stainless steel probe, set for absorbance and transmittance measurements of liquid samples, and a FLEX spectrometer (UV-Vis-NIR configured). Find out more about our pre-configured packages for UV-Vis-NIR absorbance and transmittance measurements.