Optical density and absorbance are frequently used interchangeably in laboratory settings, but they are not strictly the same. Absorbance describes the loss of light intensity as it passes through a clear solution due solely to molecules absorbing the light photons. Optical density (OD), conversely, is a broader measurement that accounts for the total reduction in light intensity, including molecular absorption and light lost to scattering or reflection.
What Absorbance Truly Measures
Absorbance (\(A\)) is a quantitative measure of how much light is stopped by a homogenous sample at a specific wavelength. This measurement is rooted in the Beer-Lambert Law, a principle that establishes a direct, linear relationship between light absorption and the properties of the substance. This law assumes that all light attenuation is caused by molecules chemically absorbing the energy of the incoming photons.
Absorbance relies on three factors that influence the amount of light absorbed. The concentration of the absorbing substance directly affects the measurement, as more molecules mean more targets for the light to interact with. Second, the path length, the distance the light travels through the sample, also influences the reading, as a longer path provides more opportunity for molecular interaction. Finally, molar absorptivity (or the molar extinction coefficient) is a unique property specific to each compound, representing how strongly that substance absorbs light at a given wavelength.
Absorbance measurements require an ideal environment where the solution is perfectly clear and non-scattering. This ideal relationship allows scientists to calculate the exact concentration of a purified compound in a solution by measuring its absorbance.
How Optical Density Differs in Practice
Optical Density (OD) is a practical metric used to quantify the total attenuation of light as it passes through a sample, especially when that sample is not perfectly clear. Its physical meaning is far more inclusive than Absorbance, accounting for all phenomena that impede the light beam. OD represents light lost due to absorption, reflection, and, most importantly, light scattering.
The distinction is most apparent in biological applications, such as estimating the concentration of cells in a liquid culture like bacteria or yeast. Researchers often measure the OD at 600 nanometers (OD600) to monitor microbial growth. At this wavelength, the culture medium itself absorbs little light, and the bacterial cells do not significantly absorb the light either.
In this context, the OD reading primarily measures the light scattering caused by the physical presence of the cells suspended in the liquid. As the number of bacterial cells increases, the suspension becomes cloudier, or more turbid, causing more light to be deflected away from the detector. The OD600 value is therefore an indirect measure of cell density, where a higher OD indicates a greater number of cells scattering the light.
Why the Terms Are Used Interchangeably
The interchangeable use of Absorbance and Optical Density stems from the fact that in many laboratory situations, the two measurements are numerically identical. When a scientist is working with a clear solution of a purified chemical compound, the amount of light scattering or reflection is negligible. In these ideal, non-turbid solutions, the total light attenuation (OD) is essentially equal to the light lost only through molecular absorption (\(A\)).
However, the distinction becomes crucial when the sample is heterogeneous or cloudy, such as a cell suspension. When working with these turbid samples, recognizing the difference ensures the data is interpreted correctly, as the OD value is a measure of turbidity, not concentration based on chemical absorption.