Colorimeter

A colorimeter is a medical laboratory instrument used to measure the concentration of colored compounds in a solution. It works on the principle of absorption spectroscopy, specifically by determining the absorbance or transmittance of a particular wavelength of light by a solution. The absorbance is directly proportional to the concentration of the analyte, according to Beer-Lambert’s Law.

Beer-Lambert’s Law (Beer’s Law)

Beer-Lambert’s Law (also known simply as Beer’s Law) is a fundamental principle in spectroscopy that relates light absorption to the material’s properties through which the light travels. It describes how the amount of light absorbed by a substance in solution is proportional to the concentration of the absorbing substance and the path length through which the light travels.

The Formula for Beer-Lambert’s Law:

A = ε⋅c⋅l

Where:

• A = Absorbance (no units, as it’s a ratio)
• ε = Molar absorptivity (or molar extinction coefficient), which is a measure of how strongly a substance absorbs light at a particular wavelength (units: L·mol⁻¹·cm⁻¹)
• c = Concentration of the solution (units: mol/L)
• l = Path length (the distance the light travels through the solution) in centimeters (cm)

Key Points:

1. Absorbance (A): The amount of light the sample absorbs. It is dimensionless and directly measured by instruments like colorimeters or spectrophotometers.
2. Molar Absorptivity (ε): A constant that depends on the specific substance being measured and the wavelength of light used. A high ε means the substance absorbs light very well at that wavelength.
3. Concentration (c): The concentration of the solute in the solution. The higher the concentration, the more light the solution absorbs.
4. Path Length (l): The distance the light travels through the sample (usually the width of the cuvette). The longer the path, the more absorption occurs.

Basic Components of a Colorimeter:

1. Light Source: A tungsten filament lamp or LED typically provides visible light. Different filters can be applied to select specific wavelengths suitable for the measurement.
2. Monochromator/Filters: These isolate the desired wavelength of light absorbed by the solution. In a colorimeter, colored filters or prisms are commonly used.
3. Sample Holder/Cuvette: This transparent container, often made of glass or plastic, holds the liquid sample. The cuvette must be clean and optically transparent for accurate readings.
4. Detector: A photodiode or photodetector is placed after the sample to detect the intensity of light that passes through the solution. The detector converts the light into an electrical signal that can be quantified.
5. Display/Readout: The output from the detector is processed and displayed, usually as absorbance or transmittance values.

Working Principle of a Colorimeter:

1. Light Emission: The colorimeter emits light, which passes through the selected filter.
2. Light Absorption: The filtered light is then directed toward the sample in the cuvette. Some of the light is absorbed by the colored compounds in the solution, while the rest passes through.
3. Measurement of Transmittance/Absorbance: The light passing through the solution hits the detector, where its intensity is compared to the intensity of the light emitted by the source.
4. Data Calculation: The difference in the intensity of the light before and after it passes through the sample provides information on the concentration of the analyte. This is displayed as the percentage of light transmitted (transmittance) or absorbed (absorbance).

Applications of Colorimetry in the Medical Laboratory:

1. Biochemical Analysis: Colorimeters are widely used for measuring concentrations of blood, serum, or urine substances such as glucose, proteins, and lipids. Various reagents react with these substances, forming a colored complex whose concentration can be quantified using a colorimeter.
2. Haemoglobin Estimation: Colorimeters are often used to measure haemoglobin concentration in blood, which is essential for diagnosing anaemia.
3. Drug Testing: In pharmacology, colorimetry helps monitor drug concentration in body fluids, ensuring proper therapeutic dosing.
4. Clinical Enzyme Assays: Colorimetric methods are used to measure enzyme activity by detecting the change in absorbance due to the formation of a colored product.
5. Water and Food Testing: In environmental and food testing laboratories, colorimetry can determine contamination levels, like nitrite concentration in water or food.

Advantages of Colorimetry:

• Simple and Quick: It provides a fast method for determining concentrations of substances in biological samples.
• Cost-Effective: The instrument is relatively inexpensive and easy to operate, making it accessible for routine tests.
• Non-Destructive: The sample remains intact for further analysis, if necessary.

Limitations of Colorimetry:

• Low Sensitivity: Colorimeters are less sensitive than advanced instruments like spectrophotometers. They are limited to visible light measurements.
• Interference: Multiple colored compounds in the sample can interfere with the readings, making the results less accurate.
• Wavelength Limitation: Colorimeters use filters, and therefore, only specific wavelengths can be selected for analysis, which limits its applicability in certain tests that require UV or infrared light.

References

1. Skoog, D. A., Holler, F. J., & Crouch, S. R. (2017). Principles of Instrumental Analysis (7th ed.).
2. Wilson, K., & Walker, J. (2010). Principles and Techniques of Biochemistry and Molecular Biology (7th ed.). Cambridge University Press.
3. Burtis, C. A., & Bruns, D. E. (2019). Tietz Textbook of Clinical Chemistry and Molecular Diagnostics (6th ed.). Elsevier.