Principle and Calibration of Ultraviolet and Visible Absorption Spectrophotometry
Principle of UV-Vis Spectrophotometry
The fundamental principle of UV-Vis spectrophotometry is based on the absorption of ultraviolet (190–400 nm) and visible (400–800 nm) light by molecules. When a molecule absorbs light, electrons are promoted from a lower energy level to a higher energy level. The amount of light absorbed at a specific wavelength is directly related to the concentration of the absorbing species in the sample, as described by the Beer-Lambert Law:
A = ε × l × c
Where:
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A is the absorbance (unitless),
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ε is the molar absorptivity (L·mol⁻¹·cm⁻¹),
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l is the path length of the sample cell (cm),
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c is the concentration of the analyte (mol·L⁻¹)
This relationship allows for the quantitative determination of analyte concentrations in a solution.
Calibration of UV-Vis Spectrophotometers
Regular calibration of UV-Vis spectrophotometers is essential to ensure accurate and reliable measurements. Calibration involves verifying and adjusting various instrument parameters using standard reference materials. Key calibration procedures include:
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Wavelength Accuracy: Ensures the spectrophotometer accurately measures specific wavelengths. This is typically verified using standard solutions like holmium oxide or didymium filters, which have known absorbance peaks at specific wavelengths.
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Photometric Accuracy (Absorbance Accuracy): Assesses the instrument’s ability to measure absorbance accurately.
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Stray Light: Evaluates the instrument’s ability to minimize light of unintended wavelengths reaching the detector. Solutions like sodium iodide or potassium chloride are used to assess stray light levels.
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Resolution Power: Determines the instrument’s ability to distinguish between two closely spaced absorbance peaks. This is often tested using a toluene in hexane solution, measuring the ratio of absorbance at specific wavelengths.
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Linearity: Checks the instrument’s response over a range of concentrations to ensure it follows the Beer-Lambert Law. This involves measuring absorbance of standard solutions at varying concentrations.
Calibration should be performed regularly, with the frequency depending on the instrument’s usage and the laboratory’s quality assurance protocols. Adhering to standard operating procedures (SOPs) and guidelines from pharmacopeias like USP, BP, and IP ensures the reliability of analytical results.