A colorimeter is an analytical instrument used widely in chemistry and biology laboratories to determine the concentration of a specific colored compound within a liquid solution. It functions by measuring the intensity of the color, which is directly related to the amount of substance present. The device works by passing a controlled beam of light through the sample and quantifying how much of that light the solution absorbs. This measurement of light absorption is then mathematically translated into a concentration value for the dissolved substance.
The Science Behind the Measurement
The fundamental principle governing the colorimeter’s operation is the Beer-Lambert Law, which establishes a quantitative relationship between light absorption and concentration. This law states that the amount of light absorbed is directly proportional to the concentration of the substance and the distance the light travels through the solution. Therefore, a more concentrated solution absorbs more light, meaning less light reaches the detector.
The measurement is expressed in two primary ways: Transmittance (\(T\)) and Absorbance (\(A\)). Transmittance is the ratio of the light intensity that passes through the sample (\(I\)) compared to the light intensity that initially hits the sample (\(I_0\)), often expressed as a percentage. Absorbance is a logarithmic function of Transmittance (\(A = -\log_{10}T\)).
Absorbance is the preferred value for determining concentration because it exhibits a direct, linear relationship with the concentration of the dissolved substance. This linearity, represented by the equation \(A = \epsilon cl\), allows a simple light reading to be converted into a precise concentration value. In this formula, \(\epsilon\) is the molar absorptivity, a constant unique to the substance and wavelength, and \(l\) is the fixed path length of the sample container.
The Path of Light Inside the Instrument
The colorimeter employs a sequence of specialized components to ensure accurate and repeatable measurements of light absorption. The process begins with a stable light source, often a low-voltage incandescent lamp or a light-emitting diode (LED), which emits a broad spectrum of light. This light must be consistent to serve as the reference intensity (\(I_0\)) for the measurement.
The next step involves a crucial component known as the wavelength selector, typically a colored filter. Since the dissolved substance only absorbs light effectively at certain wavelengths, this filter isolates a narrow band of light, usually the complementary color to the solution’s color, to maximize absorption. For example, a blue solution will absorb orange or red light most strongly, so a red filter would be selected for the analysis.
The filtered, monochromatic light then passes through the sample, which is contained in a small, transparent vessel called a cuvette. The cuvette is designed with a precise, fixed path length, commonly 1 centimeter. This fixed distance ensures that path length (\(l\)) remains constant in the Beer-Lambert Law equation, isolating changes in light absorption solely to changes in concentration.
Finally, the light that successfully passes through the sample (\(I\)) strikes a photoelectric detector, such as a photocell or photodiode. This sensor measures the intensity of the transmitted light and converts the light energy into a proportional electrical signal. The colorimeter’s internal electronics then use this electrical signal, along with the initial light intensity, to calculate the sample’s Transmittance and Absorbance.
Converting Light Readings into Concentration
The raw absorbance reading is a measure of light attenuation, not concentration, and requires conversion into a usable concentration value. This conversion relies on calibration, which involves creating a standard curve. To begin, a series of standard solutions must be prepared, where the exact concentration of the colored substance is already known.
The absorbance of each known standard solution is measured individually using the colorimeter. Before measuring the standards, a “blank” solution is used to set the instrument’s zero point; this solution contains all reagents and solvent except the colored substance. The blank accounts for any background absorption from the solvent or container, ensuring the final reading is only for the substance of interest.
The measured absorbance values are plotted against their known concentrations. This generates the standard curve, which should ideally show a straight line due to the linear relationship established by the Beer-Lambert Law. Once established, the absorbance of the unknown sample is measured and compared to the plotted line.
The concentration of the unknown sample is determined by finding its absorbance value on the vertical axis and tracing a line horizontally to the standard curve, then dropping a vertical line down to the horizontal concentration axis. This process, called interpolation, translates the measured light property (absorbance) into the desired chemical property (concentration). The colorimeter uses this mathematically defined relationship to report the final concentration of the substance.