Understanding Absorbance
Absorbance quantifies how much light a substance takes in when light passes through it. When incident light shines on a sample, some light is absorbed, while the rest is transmitted. This measurement helps scientists understand how much light energy a sample retains. Absorbance is calculated as the common logarithm of the ratio of incident light intensity to transmitted light intensity. Since it compares two quantities of the same type, absorbance is a dimensionless quantity, meaning it has no units.
Commonly Used Terms for Absorbance
Despite being technically unitless, terms like “Absorbance Units” (AU) or “Optical Density” (OD) are frequently encountered in scientific practice. These terms are common in fields relying on light-based measurements, such as ultraviolet-visible (UV-Vis) spectroscopy (analyzing light absorption at different wavelengths) and high-performance liquid chromatography (HPLC) for separating and quantifying mixture components. While widely used, AU and OD are not true physical units. Organizations like the International Union of Pure and Applied Chemistry (IUPAC) often discourage their use, advocating for greater clarity and consistency. “Optical Density” is an older synonym for absorbance, but its historical use also included light scattering, which can lead to confusion.
The Beer-Lambert Law
The relationship between absorbance and the properties of the absorbing substance is described by the Beer-Lambert Law, expressed as A = εlc. In this equation, ‘A’ represents absorbance, a unitless value. ‘c’ denotes the concentration of the light-absorbing substance in solution, typically measured in moles per liter (mol/L). ‘l’ refers to the path length light travels through the sample, commonly expressed in centimeters (cm).
‘ε’ (epsilon) is the molar absorptivity or molar extinction coefficient. This coefficient is specific to each substance at a given wavelength and temperature, and it is the only term in the Beer-Lambert Law that carries units. Molar absorptivity commonly has units of liters per mole per centimeter (L mol⁻¹ cm⁻¹), or equivalently M⁻¹cm⁻¹. When these units are multiplied by the concentration (mol/L) and the path length (cm), the units effectively cancel out (L/mol·cm mol/L cm), resulting in a unitless value for absorbance, reinforcing its dimensionless nature.
Practical Applications of Absorbance
Absorbance measurement provides scientists with a powerful tool for quantitative analysis across various disciplines. In chemistry and biology, it is routinely used to determine the concentration of substances in solution, such as DNA, proteins, or specific chemical compounds. For example, protein concentration can be estimated by measuring its absorbance at a specific wavelength, often 280 nanometers.
Beyond concentration determination, absorbance also monitors chemical reaction progress by observing changes in reactant or product absorption over time. It plays a role in environmental science for assessing water quality by detecting pollutants. The utility of absorbance in providing precise quantitative information makes it an indispensable technique in research, diagnostics, and industrial processes.