An absorbance assay is a foundational laboratory method used to determine the concentration of a substance in a solution, with applications across biochemistry, environmental analysis, and pharmaceutical quality control. The assay’s function is to measure a substance by assessing how it interacts with light. This provides a straightforward way to quantify materials like proteins, DNA, and various chemical compounds.
The Principle of Spectrophotometry
An absorbance assay operates on the principle of spectrophotometry, which measures how much light a chemical substance absorbs. Every compound absorbs and transmits light over a specific range of wavelengths. When a beam of light passes through a sample solution, the molecules within it absorb some of that light energy. This reduces the intensity of the light that emerges on the other side.
This relationship is described by the Beer-Lambert Law, which states there is a linear connection between a solution’s absorbance and the concentration of the substance dissolved in it. To measure this, scientists use an instrument called a spectrophotometer. A spectrophotometer contains a light source, a monochromator to select a specific wavelength, a holder for the sample (a cuvette), and a detector. The instrument compares the initial light intensity to the final intensity to calculate the absorbance value.
Conducting an Absorbance Assay
The execution of an absorbance assay involves a series of precise steps. The process begins with preparing the sample, which may involve dissolving it in a solvent or mixing it with reagents that produce a color change. An example is the Bradford assay, where a dye reacts with proteins to create a blue color whose intensity is proportional to the protein concentration. This colorimetric change allows the spectrophotometer to measure substances that might not absorb light strongly on their own.
Calibrating the spectrophotometer with a “blank” is a required step. A blank is a solution containing everything found in the sample except for the substance being measured, such as the solvent and any reagents. By first measuring the blank, the instrument can be set to zero, ignoring any background absorbance from the cuvette or the solution itself. This ensures the final reading reflects only the absorbance of the target substance.
To determine an unknown sample’s concentration, a standard curve is created by preparing a series of solutions with known concentrations. The absorbance of each standard is measured and plotted on a graph with absorbance on the y-axis and concentration on the x-axis, forming the standard curve. The absorbance of the unknown sample is then measured, and its concentration is found where its absorbance value intersects with this curve.
Common Applications
In molecular biology and biochemistry, absorbance assays are routinely used to quantify the concentration of proteins. Assays like the Bradford or BCA (bicinchoninic acid) assay are standard procedures for studying protein function and expression. These methods rely on a reagent that changes color in the presence of protein, and the resulting absorbance is measured to determine its concentration.
Absorbance assays are also used for measuring nucleic acid purity and concentration. DNA and RNA naturally absorb ultraviolet (UV) light at 260 nanometers (nm), which allows researchers to calculate their concentration. By comparing this reading to the absorbance at 280 nm, the wavelength at which proteins absorb light, they can also assess the purity of the nucleic acid sample.
These assays are also employed to study enzyme kinetics. Scientists monitor an enzymatic reaction’s progress by measuring the change in absorbance over time. This is achieved by tracking the appearance of a product or the disappearance of a substrate that absorbs light at a particular wavelength. In clinical diagnostics, the assays analyze components in biological fluids, like measuring compounds in blood or urine to help diagnose diseases.
Variables Affecting Assay Results
Several factors can influence the accuracy and reliability of an absorbance assay. To achieve accurate results, the following variables must be controlled:
- Wavelength selection: For any substance, there is a specific wavelength at which it absorbs light most strongly, known as its maximum absorbance (λmax). Using this wavelength provides the highest sensitivity and yields the most accurate measurements.
- Chemical environment: The sample’s pH and temperature can affect results. Changes in these conditions can alter a molecule’s structure, which in turn changes its light-absorbing properties. For instance, a protein might unfold at high temperatures, leading to an incorrect concentration reading.
- Interfering substances: Contaminants in the sample that absorb light at the same wavelength as the target substance will lead to an artificially high absorbance reading and are a potential source of error.
- Handling errors: Simple mistakes can introduce variability. Scratches, fingerprints, or air bubbles on or in the cuvette can scatter or absorb light, interfering with the measurement.