A spectrophotometer measures the amount of light a substance absorbs. It operates by passing a light beam through a sample and quantifying the light taken in. This provides insights into the sample’s composition and concentration, performing quantitative analysis across various scientific disciplines.
Understanding Absorbance
Absorbance is a quantitative measure of how much light a substance “takes in” rather than allowing to pass through. This contrasts with transmittance, which indicates the amount of light that successfully passes through a sample. Measuring absorbance allows scientists to determine the concentration of specific compounds within a solution. As the concentration of an absorbing substance increases, more light is absorbed, making absorbance a direct indicator of concentration.
The Spectrophotometer’s Essential Parts
A spectrophotometer relies on several key components. A light source, such as a tungsten or deuterium lamp, provides the initial beam of radiation. This light then enters a monochromator, which separates the light into its individual wavelengths and selects a specific, narrow band to pass through. The selected light travels through a cuvette, which holds the liquid sample. After passing through the sample, the light reaches a detector, such as a photodiode, which measures the intensity of the transmitted light, and a display system then presents the measured absorbance values to the user.
The Measurement Sequence
The process of measuring absorbance with a spectrophotometer begins by directing light from the source through the monochromator to select a specific wavelength. Before measuring the sample, the instrument is calibrated using a “blank” solution. This blank accounts for any background absorption from the solvent or cuvette. The light passes through this blank, and the instrument is adjusted to read zero absorbance, effectively setting a reference point.
After calibration, the light of the same selected wavelength is directed through the actual sample contained in a cuvette. As the light passes through the sample, some of it is absorbed by the molecules present. The detector then measures the intensity of the light that successfully transmits through the sample. The spectrophotometer’s internal system compares this transmitted light intensity to the initial light intensity (or the intensity measured through the blank). Based on this comparison, the instrument calculates the absorbance value, which quantifies how much light was taken in by the sample at that specific wavelength.
The Beer-Lambert Principle
The Beer-Lambert Law is the fundamental principle underlying spectrophotometric measurements. This law establishes a direct and proportional relationship between the absorbance of a solution and two factors: the concentration of the absorbing substance and the path length of the light through the sample. If the concentration of a substance doubles, its absorbance will also double, assuming the same path length. A longer path length through the sample will similarly result in higher absorbance.
The Beer-Lambert Law enables scientists to determine the concentration of an unknown substance by measuring its absorbance. Its practical application is straightforward: a higher absorbance reading indicates a greater concentration of the light-absorbing compound. This principle is widely applied in various fields to quantify substances, from analyzing DNA and proteins in biological research to monitoring environmental pollutants.