A colorimeter is an analytical instrument that measures the intensity of light absorbed by a colored compound in a liquid solution. By shining a beam of light at a specific wavelength through the sample, the device quantifies the light absorbance. This measurement directly relates to the concentration of the colored substance within the solution. The technique is widely used in chemistry and biology laboratories, environmental testing, and clinical diagnostics for analyzing blood and urine samples.
Preparing the Colorimeter and Test Solutions
Before measurement, preparation of the instrument and samples is necessary. This involves selecting the correct wavelength filter, which is determined by the color of the solution being analyzed. The filter should isolate the color of light that the compound absorbs most strongly, known as the maximum absorbance. For instance, a blue solution absorbs light most effectively in the orange-red region, requiring a filter isolating a wavelength around 600-650 nanometers.
Sample handling requires the use of cuvettes, which hold the liquid in the colorimeter’s light path. Cuvettes must be cleaned thoroughly and handled only by the frosted sides to prevent fingerprints from interfering with the light path. Any smudge or scratch on the clear sides can scatter or absorb light, leading to an incorrect reading. The cuvette must be filled high enough to intersect the light beam completely, but not so high as to spill into the instrument.
The preparation of the “blank” solution is essential, as it serves as the reference point for the measurement. This blank usually consists of the solvent used, or all reagents except the substance being analyzed. The blank solution calibrates the instrument by accounting for any background absorbance from the solvent or the cuvette itself. Subsequent sample readings will then reflect only the light absorbed by the colored compound of interest.
Performing the Measurement
The measurement process begins by turning on the colorimeter and allowing it to warm up, typically for around 15 minutes, until the instrument stabilizes. This warm-up time ensures a consistent light source intensity and detector response for reliable measurements. Once the device is stable, the selected wavelength filter is positioned to isolate the light appropriate for the sample.
Calibration, or “zeroing,” is performed next by inserting the prepared blank solution into the sample holder. The colorimeter is adjusted to read zero absorbance or 100% transmittance. This sets the baseline by instructing the instrument to disregard light absorption caused by the cuvette and the solvent. The blank cuvette is then removed, and the sample measurement process can begin.
Each prepared sample cuvette is carefully inserted into the sample holder, ensuring the clear sides are aligned with the light path. Once the sample is in place, the instrument measures the light passing through and displays the result as an absorbance value. This reading is recorded before moving on to the next sample. Maintaining consistent alignment for each cuvette placement is important for minimizing measurement variability.
Translating Measurements into Results
The absorbance value displayed by the colorimeter is not the concentration itself. Interpretation relies on the Beer-Lambert Law, which states that the concentration of a substance is directly proportional to the amount of light it absorbs. In simpler terms, a darker solution absorbs more light, indicating a higher concentration of the colored compound.
To translate the measured absorbance into a specific concentration, a standard curve is first generated. This involves preparing a series of solutions with known concentrations of the substance and measuring the absorbance of each. The measured absorbance values are then plotted on a graph against their corresponding concentrations, creating the standard curve. This curve acts as a reference for the compound under the specific measurement conditions.
The absorbance reading from the unknown sample is located on the y-axis of the standard curve. By tracing a line from this absorbance value across to the plotted curve and down to the x-axis, the corresponding concentration of the unknown sample is determined. This method provides a specific concentration that can be used for research, quality control, or diagnostic purposes.