Colorimetric Analysis Quiz

Principles of Colorimetric Analysis

• Colorimetric analysis is based on the principle that many substances in solution can be colored or produce a colored derivative.
• The intensity of color in a solution is related to the amount of substance present, with higher concentrations appearing darker in color.
• Colored substances absorb light at specific wavelengths in the visible spectrum, with pale colored solutions absorbing only a small amount of light and dark colored solutions absorbing a lot of light.
• The visible light spectrum ranges from about 400 nm to 700 nm, with wavelengths shorter than 400 nm being ultraviolet and longer than 700 nm being infrared.
• A substance's ability to selectively absorb certain wavelengths of light is determined by its molecular and atomic structures.
• The Beer-Lambert law states that the amount of light absorbed by a colored solution is directly proportional to the concentration of the solution and the length of the light path through it.
• Colorimetric tests use the Beer-Lambert law to determine the concentration of a substance in an unknown solution by comparing it to a standard.
• The amount of light absorbed (optical density or OD) is dependent on the light path distance and wavelength used.
• Colorimeters and spectrophotometers are used to measure absorbance, with colorimeters using colored filters to cover wavelength ranges and spectrophotometers using a diffraction grating or prism to disperse white light into a continuous spectrum.
• The Walden Precision Apparatus (WPA) CO700D medical colorimeter has a light source, filters, photosensitive detector system, and cells (cuvettes) to hold solutions.
• To ensure accurate results, solutions used in colorimetric tests must be homogenous, the substance being measured should not react with the solvent, and a reagent blank must be used to correct for any absorption of light by solvents.
• The Beer-Lambert law can be applied to most clinical chemistry quantitative tests, as long as reliable chemicals and good technique are used to prepare solutions.