Spectrophotometry is a fundamental analytical technique employed across biology, chemistry, and various industries to measure how light interacts with matter. The core principle involves shining a specific wavelength of light through a sample and quantifying the amount of light the sample retains or lets pass through. This measurement is the foundation for determining the identity and amount of a substance in a solution. The central concept for quantitative analysis is absorbance, which provides a direct link to the concentration of the substance being studied.
Defining Absorbance and Transmittance
When a beam of light, called incident light, hits a solution, several interactions can occur, including reflection, scattering, and retention by the molecules within the sample. Transmittance (\(T\)) is defined as the fraction of the incident light that successfully passes through the sample and reaches a detector. It is calculated as the ratio of the transmitted light intensity (\(I\)) to the incident light intensity (\(I_0\)), and is often expressed as a percentage (0% to 100%).
Absorbance (\(A\)), conversely, quantifies the amount of light retained or blocked by the sample, primarily due to the absorption of photons by the molecules of interest. Unlike transmittance, which relates linearly to light intensity, absorbance is defined on a logarithmic scale: it is the negative logarithm (base 10) of the transmittance (\(A = -\log_{10}T\)). This logarithmic formulation is used for analytical purposes.
This mathematical relationship means that as the amount of light passing through the sample decreases, the absorbance value increases. For example, a transmittance of 10% corresponds to an absorbance of \(1.0\), while 1% corresponds to an absorbance of \(2.0\).
The practical advantage of using absorbance is that it directly relates to the concentration of the absorbing substance, unlike transmittance. This linear proportionality allows spectrophotometry to be used as a reliable quantitative tool.
The Cornerstone Principle: The Beer-Lambert Law
The mathematical relationship linking measured absorbance to the concentration of an analyte is formalized in the Beer-Lambert Law, expressed by the equation \(A = \epsilon lc\). This law establishes a direct, linear correlation between the absorbance (\(A\)) of a solution and the amount of the absorbing substance it contains.
The variable \(\epsilon\), known as the molar absorptivity or molar extinction coefficient, is a unique constant for a specific substance at a particular wavelength. It measures how strongly that compound absorbs light at the selected wavelength, and its units are typically \(L \cdot mol^{-1} \cdot cm^{-1}\).
The term \(l\) represents the path length, the distance the light travels through the solution, typically measured in centimeters. In most laboratory settings, the sample is held in a cuvette, which has a standard path length of \(1.0\) cm. Since the path length is usually kept constant, it simplifies quantitative analysis.
Finally, \(c\) is the concentration of the absorbing substance, usually expressed in moles per liter (\(M\)). The Beer-Lambert Law demonstrates that if \(\epsilon\) and \(l\) are held constant, doubling the concentration will double the measured absorbance. This linear dependence is why the law serves as the foundation for determining unknown concentrations.
The Measurement Process: Components of a Spectrophotometer
The measurement of absorbance is performed using a spectrophotometer, an instrument designed to precisely control and measure the interaction of light with a sample. The process begins with the light source, which provides a stable beam of light (e.g., a tungsten lamp for the visible spectrum or a deuterium lamp for the ultraviolet range).
The light then passes through a monochromator, a system containing a prism or a diffraction grating that separates the light into its individual wavelengths. This component allows the user to select a narrow band of light at a specific wavelength, often the \(\lambda_{max}\) where the sample absorbs light most strongly.
The selected monochromatic light is then directed through the sample holder, typically a cuvette. The cuvette holds the liquid sample and is positioned so the light beam travels a defined path length through the solution. After passing through the sample, the transmitted light reaches a detector, which converts the light energy into an electrical signal.
Before measuring the sample, a necessary operational step called “blanking” is performed to zero the instrument. This involves placing a “blank” solution—containing the solvent and any reagents but not the absorbing substance—into the cuvette. The spectrophotometer measures the light intensity passing through the blank, setting this value as the \(100\%\) transmittance mark. This step excludes any absorbance caused by the solvent or the cuvette itself from the final reading. The detector then measures the transmitted intensity of the sample, and the instrument calculates the final absorbance value.
Practical Applications of Absorbance
The ability to precisely measure absorbance has made spectrophotometry an indispensable tool across numerous scientific disciplines. In molecular biology, a common application is the quantification of nucleic acids like DNA and RNA, which naturally absorb ultraviolet light at 260 nm. Measuring the absorbance at this specific wavelength allows researchers to determine the concentration of genetic material in a solution.
Another widespread use is monitoring the rate of biochemical reactions, known as enzyme kinetics. Many assays are designed so the reaction product is a colored molecule that absorbs light at a specific wavelength. By measuring the change in absorbance over time, scientists can track the formation of the product and calculate the speed of the enzyme reaction.
In industrial settings, absorbance measurements are used for quality control and process monitoring. Pharmaceutical companies use the technique to quantify active ingredients in drug formulations, ensuring compliance with regulatory standards. The food and beverage industry uses it to assess the concentration of additives, pigments, and nutrients. It is also used to monitor the growth of microorganisms in cultures, as increasing cell density leads to an apparent increase in absorbance.