A spectrophotometer is a scientific instrument designed to measure how much light a chemical substance absorbs or transmits at specific wavelengths. This measurement provides both qualitative and quantitative data about the sample’s composition and concentration. By precisely controlling the light that passes through a sample and measuring the resulting intensity, the device reveals how matter interacts with electromagnetic radiation. The technology is fundamental to analysis in fields ranging from biology and chemistry to quality control in industry.
Essential Components and Their Roles
The process begins with a stable, high-intensity light source, which is typically a tungsten lamp for the visible spectrum or a deuterium lamp for the ultraviolet range. This source produces a broad spectrum of light containing many different wavelengths, similar to white light. The light beam then travels to a component called the monochromator, which is responsible for isolating a single, narrow band of wavelengths.
Inside the monochromator, a prism or a diffraction grating separates the polychromatic light into its component colors. A mechanical slit then selects only the desired wavelength to pass through, ensuring the sample is exposed to monochromatic light. The absorption properties of a substance are specific to each wavelength, making this isolation necessary.
The precisely selected light beam next enters the sample compartment, where the solution is held in a transparent container known as a cuvette. The cuvette has a fixed path length, usually one centimeter. As the light passes through the solution, the chemical molecules within the sample interact with and absorb some of the light energy.
Finally, the remaining light strikes the detector. This detector is typically a photodiode or a photomultiplier tube that converts the light energy into a measurable electrical current. The magnitude of this current is directly proportional to the intensity of the transmitted light, allowing the instrument to precisely quantify how much light made it through the sample.
Measuring Light: Absorption and Transmission
The spectrophotometer’s core function is to compare the initial intensity of the light beam before it enters the sample to the intensity of the light that exits the sample. This comparison yields two fundamental measurements: Transmittance and Absorbance. Transmittance (\(T\)) is defined as the fraction of the incident light that successfully passes through the sample, often expressed as a percentage.
If a sample is completely transparent to the selected wavelength, the light intensity remains nearly unchanged, resulting in high transmittance. Conversely, if the sample strongly absorbs the light, the transmitted intensity is low, resulting in low transmittance. The value of absorbance (\(A\)) is mathematically derived from transmittance, specifically as the negative logarithm of transmittance.
This logarithmic relationship means that as the amount of light transmitted decreases, the absorbance value increases exponentially. Spectrophotometers measure absorbance because this value offers a linear relationship with the concentration of the absorbing substance. By quantifying the light that disappears in the sample, the instrument provides a direct metric for the amount of substance present.
Translating Data into Concentration
The measured absorbance value is converted into a meaningful concentration using a physical principle known as the Beer-Lambert Law. This law establishes a direct, linear proportionality between the absorbance of a solution and the concentration of the absorbing species. The mathematical expression of this law is \(A = \epsilon cl\), where \(A\) is the measured absorbance.
The equation includes two constant values for a given experiment: \(l\), the path length of the light through the cuvette, and \(\epsilon\), the molar absorptivity. Molar absorptivity is a constant unique to a specific substance at a specific wavelength, describing how strongly that molecule absorbs light.
Since both the path length and the molar absorptivity are held constant, the equation simplifies to show that absorbance is directly proportional to concentration (\(c\)). By measuring the absorbance of a sample and knowing the values for the other two constants, the spectrophotometer can precisely calculate the unknown concentration of the substance.