Absorbance & Transmittance
Absorbance (A) and Transmittance (T) are measurements used in molecular spectroscopy. Molecular spectroscopy measures the fraction of light that a transparent and finite-length sample absorbs or transmits over a given wavelength range.
This technique is useful for determining the identity and concentration of an unknown absorbing specie within a sample.
Common questions
How does molecular absorption occur?
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.
Figure 1 – Molecular absorption process between the ground and excited state.
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 neglects reflection losses, the incident intensity is equal to the transmitted intensity.
How can you use Absorbance for Quantitative Analysis?
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 I₀ and Iᴛ 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.
Figure 2 – Absorption of radiant energy that goes through a transparent sample, with a finite path length.
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.
How is Transmittance related to Absorption?
The quantitative absorption of radiant energy by an 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.
How are the Absorption properties of a sample represented?
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.
Typical Setups for Absorbance & Transmittance
The setups supplied by Sarspec for a typical absorbance and transmittance measurement work by transporting the light produced by a deuterium and/or tungsten-halogen light source (LS-DW, LS-DWHP, or LS-W) through an optical fiber to a sample holder, which might be a cuvette holder, flow cell, or transmittance probe. A second optical fiber transports the light from the sample holder to the spectrometer, which detects and records the intensity of transmitted light in real time.