The measurement of light absorption is a fundamental method in chemistry and biology used to quantify specific substances in a solution. When light passes through a medium, some energy is taken up by the molecules within that substance. This phenomenon, known as absorbance, allows researchers to indirectly determine the concentration of a compound. The degree to which a sample interacts with light is a unique physical characteristic.
Understanding the Core Concept
Absorbance (A) describes the amount of light retained by a sample. It is mathematically related to transmittance (T), which is the amount of light that successfully passes through the sample. Transmittance is typically expressed as a percentage of the initial light intensity that reaches the detector. As absorbance increases, transmittance decreases.
Absorbance is a logarithmic value derived from the ratio of the initial light intensity to the transmitted light intensity. This means it does not increase linearly like transmittance. For example, a solution absorbing 90% of light has an absorbance of 1.0, while one absorbing 99% has an absorbance of 2.0. This logarithmic scale compresses a wide range of concentration changes into a manageable numerical range.
The Tool for Measurement
The instrument designed to quantify this light interaction is the spectrophotometer, sometimes referred to as a colorimeter. It operates by analyzing how a sample affects a beam of light. The process begins with a light source generating electromagnetic radiation, which then passes through a monochromator or filter to isolate a narrow band of wavelengths.
Selecting a specific wavelength ensures the measurement focuses on the wavelength most effectively absorbed by the target molecule. The isolated beam travels through the sample, held in a specialized container called a cuvette. The remaining light strikes a detector, which converts the energy into an electrical signal displayed as an absorbance value.
The Mathematics Governing Absorbance
The quantitative relationship between measured light absorption and solution properties is described by a principle of direct proportionality. Absorbance (A) is directly proportional to two primary factors: the concentration of the absorbing substance and the distance the light travels through the sample.
The proportional factors include the concentration ($c$) of the solute and the path length ($l$), which is the internal width of the cuvette (usually 1 centimeter). The third factor is the molar absorptivity ($\epsilon$), a constant specific to the substance and the chosen wavelength, representing the inherent efficiency with which a molecule absorbs light.
Since the path length and molar absorptivity are held constant, any change in measured absorbance must be due to a change in concentration. To translate an absorbance reading into a concentration value, scientists first generate a standard curve. This is done by measuring the absorbance of several solutions with known concentrations, creating a reference line to calculate the concentration of any unknown sample.
Practical Steps for Taking a Reading
Accurate measurement begins with selecting the optimal wavelength ($\lambda$ max) for the target substance. This is the specific wavelength where the compound absorbs the most light, maximizing the sensitivity of the measurement. This step is performed by scanning the sample across a range of wavelengths to identify the peak absorption point.
Before measuring samples, the instrument must be “blanked” or “zeroed” to eliminate background interference. This involves placing a reference solution—containing all components except the substance being measured—into the spectrophotometer. Measuring the blank and setting the absorbance to zero subtracts any light absorption caused by the solvent or cuvette.
Proper handling of the cuvette is also necessary to ensure the light beam passes cleanly through the sample. Cuvettes should only be handled by the frosted sides to prevent fingerprints from smudging the clear optical surfaces. Residue or scratches on the clear sides can scatter or absorb light, leading to an inaccurate absorbance reading.