Absorption is measured by determining the reduction in intensity of a light beam after it passes through a substance. This measurement is typically done using a spectrophotometer or absorbance reader.
Here's a breakdown of the process:
The Traditional Cuvette Method
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Sample Preparation: A sample containing the substance of interest (analyte) is dissolved in a suitable solvent. The concentration of the analyte needs to be known, or at least consistent for comparative analyses.
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Cuvette Placement: The solution is placed in a cuvette, a small rectangular container with transparent walls that is designed to hold liquids in the light path of the measuring instrument.
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Light Source: A light source emits a beam of light with a known intensity (I₀). The light source might be a lamp that emits across a range of wavelengths (like a spectrophotometer) or a light source that emits light at a specific wavelength (like a colorimeter).
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Light Transmission Through Sample: The light beam passes through the cuvette containing the sample.
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Light Detection: A detector on the other side of the cuvette measures the intensity of the light that passes through the sample (I).
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Absorbance Calculation: The absorbance (A) is calculated using the following formula, derived from the Beer-Lambert Law:
A = -log₁₀ (I / I₀)
Where:
- A = Absorbance (unitless)
- I = Intensity of light transmitted through the sample
- I₀ = Intensity of the incident light (light before it hits the sample)
It's important to note that absorbance is related to transmittance (T) by the equation A = -log₁₀(T), where T = I/I₀.
Alternative Measurement Techniques
While the cuvette method is common, other techniques exist:
- Fiber Optic Probes: These probes can be dipped directly into a sample, eliminating the need for cuvettes. They are useful for in-situ measurements.
- Thin Film Analysis: Specialized instruments are used to measure the absorbance of thin films or coatings.
- Remote Sensing: Spectrometers on satellites or airplanes can measure the absorption of light by the atmosphere or the Earth's surface.
Factors Affecting Absorbance Measurements
Several factors can affect the accuracy of absorbance measurements:
- Solvent: The solvent used to dissolve the sample must be transparent at the wavelength of interest.
- Temperature: Temperature can influence the absorbance of some substances.
- Path Length: The path length of the cuvette (the distance the light travels through the sample) must be known and consistent.
- Instrument Calibration: The spectrophotometer or absorbance reader must be properly calibrated to ensure accurate readings.
- Stray Light: Stray light within the instrument can cause inaccurate absorbance readings, especially at high absorbance values.
In summary, absorption is measured by comparing the intensity of light before and after it passes through a sample, typically using a spectrophotometer and cuvette, then calculating the absorbance using the Beer-Lambert Law. Other methods exist for specific applications, and accurate measurements require careful attention to various factors.
