Spectrophotometry is a widely used technique in science for measuring how much light a chemical solution absorbs. This measurement is a key step in determining the concentration of a substance, such as a protein, DNA, or a colored compound, within a sample. The terms “Absorbance” (A) and “Optical Density” (OD) are frequently used, often interchangeably, leading to confusion about their exact relationship. While related, they describe slightly different phenomena of light interaction with matter.
Understanding Absorbance and Light Transmission
Measuring light interaction begins by shining a beam of monochromatic light, which is light of a single, specified wavelength, through a sample. When this incident light passes into the solution, some of its energy is taken up by the molecules of the substance. The light that successfully exits the sample is called transmitted light. Absorbance is a measure of the light that is held back or “trapped” by the sample, derived from the ratio of the incident light to the transmitted light. Transmittance (T) is the opposite, representing the fraction of light that passes through the sample. Absorbance and Transmittance are related through a logarithmic function: A = log(incident light / transmitted light). Since it is a ratio of light intensities, Absorbance is a unitless quantity, often reported as “Absorbance Units” (AU).
The Mathematical Relationship: The Beer-Lambert Law
The fundamental calculation connecting the measured Absorbance to the properties of the solution is known as the Beer-Lambert Law. This law establishes a direct, linear relationship between the concentration of a compound and the light absorbed by the sample. The mathematical expression of this principle is A = E l c, where A is the measured Absorbance value. E (molar absorptivity or extinction coefficient) is a constant specific to the absorbing substance at a particular wavelength, quantifying how strongly a substance absorbs light. The path length, l, is the distance the light travels through the sample, which is determined by the size of the cuvette, and is usually 1 centimeter in standard laboratory settings. The concentration of the absorbing species in the solution is represented by c, which is typically measured in moles per liter. This relationship demonstrates that if the path length and the extinction coefficient are kept constant, any increase in the sample’s concentration will result in a proportional increase in the measured Absorbance. This linear relationship is the basis for using spectrophotometry to quantify unknown concentrations of a substance.
Practical Steps for Measurement and Calculation
The practical calculation of Absorbance begins with the use of a spectrophotometer, which is the instrument used to collect the necessary light intensity data. The first step is to select the optimal wavelength, which is the specific wavelength where the substance absorbs light most strongly, providing the most sensitive measurement. Before measuring the sample, the instrument must be “zeroed” or “blanked” using a control solution that contains everything in the sample except the substance being measured. This blanking step corrects for any background absorption from the solvent or the cuvette itself by setting the Absorbance of the control solution to zero. After this preparation, the instrument directly reports the Absorbance value (A). Once this raw Absorbance value is obtained, calculating the sample’s concentration (c) is a straightforward process of rearranging the Beer-Lambert equation: c = A / (E l).
The Specific Use of Optical Density in Biological Systems
While Absorbance is the technically precise term for the light energy chemically taken up by a molecule, Optical Density (OD) is often used in a broader, more practical sense, especially in biological research. Optical Density specifically refers to the overall attenuation of light as it passes through a medium, which includes both true absorption and the effect of light scattering. Light scattering occurs when light rays hit particles larger than the light’s wavelength, such as cells or cellular debris, causing the light to deflect away from the detector. This broader definition explains why scientists measure the density of bacterial or yeast cultures using \(OD_{600}\). At the 600 nanometer wavelength, the bacterial cells in the liquid culture do not significantly absorb the light. Instead, the suspended cells scatter the light, and the resulting OD value reflects the turbidity, or cloudiness, of the culture. A higher \(OD_{600}\) reading indicates a greater number of cells, allowing researchers to monitor cell growth.