What is absorbance?
To understand the concept of absorbance, we have to consider a substance and a beam of incident light. If photons interact with the substance, the amount of light will be reduced after crossing the sample. Absorbance (A) is a measure of this reduction of light: the higher the absorbance of the substance, the lower the amount of light that passes through the substance and comes out the other side. Mathematically, absorbance is expressed as the logarithm of the ratio of incident (I0) to transmitted (I) radiant power through a sample: A = Log10(I0/I).
Absorbance is inversely proportional to transmittance: transmittance (T) is the fraction of incident light which is transmitted. If all light passes through a sample, none is absorbed, so the absorbance is zero and the transmission is 100%. On the other hand, if no light passes through the sample, the absorbance is infinite, and the percent transmission is zero.
The main physical phenomenon underlying absorbance is absorption, but in some cases, light reflection and scattering also contribute to the reduction of transmitted light.
Principle of absorbance measurements
The basic principle of absorbance measurements is described by Lambert-Beer’s law, which describes the wave propagation of radiation through a medium [1]. The absorption of light with an initial intensity I0 penetrating a diluted solution of a given substance over a given length depends on the concentration (c) of the substance, the length that has to be crossed by light (b), and the ability of the substance to interact with light.
Substances have different abilities to interact with light of a specific wavelength; generally speaking, it can be said that, if a photon interacts with a molecule of the substance of interest, it will be absorbed. As a result of this process the light intensity is attenuated after it has passed through the sample, i.e. the number of photons that appear at the end of the sample volume is reduced compared to the number of photons entering the sample. The ability of a substance to absorb light of a given wavelength is expressed as its molar extinction coefficient (also known as molar absorption coefficient or molar attenuation coefficient, ε). Taking everything into account, the common expression of Lambert-Beer’s law for the absorbance A is:
A = log (I / I0) = ε ∙ c ∙ d
Absorbance is dimensionless, but its values are usually referred to as “Absorbance units” (AU) or “Optical Density” units (OD). It is also important to note that small increases in absorbance can represent large changes in light intensity: every unit of absorbance increase represents a decrease of 10 times in the intensity of transmitted light and, for diluted samples, an increase of 10 times in the concentration of the substance of interest.
Although appearing rather straight forward, Lambert-Beer’s law is only valid under well-defined conditions and there are several challenges when applying the law in practice.
Different molecules have different absorption spectra, which gives them their characteristic colour. Accordingly, the selection of the measurement wavelength must be specified precisely [2]. Typically, one will choose a wavelength where the absorbance is maximal to obtain the highest sensitivity.
Lambert-Beer’s law idealises the processes that occurs during a transmission measurement by considering exclusively light attenuation by molecular absorption. Especially losses due to scattering are not included in the theoretical description [3]. In general, scatter deflects part of the light beam so that it is not reaching the detector at all or at least not in a straight way. This scattering may occur at any objects in the beam path, e.g. at optical components such as lenses or shutters but also particulate contaminations on surfaces or in the sample solution itself can be the origin of optical scattering. Moreover, any dielectric surface partly reflects impinging light, causing an attenuation of e.g. up to 10% for glass surfaces. Therefore, in absorbance studies the unattenuated intensity I0 is determined typically in a way that the beam path contains all elements of the actual measurement setup, including solvent and sample container, except for the actual analyte substance. This procedure guarantees that the measurement parameters are exactly the same as in the actual measurement. For classical spectrophotometers using a cuvette as a sample container, this is accomplished either by providing a separate reference channel or by two successive measurements.
In microplate absorbance readers the situation is more complex. To facilitate an absorbance measurement in any of the microplate wells the I0 measurement is performed “through the air” by default. This means, the complete sample is bypassed and the unattenuated irradiation is measured. Although widely accepted, this procedure is not suited to determine absolute concentrations with highest accuracy. Thus, it is recommended to use a well in the microtiter plate that contains the solvent only, e.g. buffer, to measure I0. In that case, the instrument takes the absorbance of that particular well as the I0 value to calculate the concentration.
For further information about the measurement of absorbance in microplates, check out our technical note “Quantitative absorbance measurements in microplates”.
Berthold Technologies manufactures instruments that cover all absorbance-based assays: filter-based absorbance readers, filter-based and monochromator-based multimode readers, and microvolume spectrophotometers.