Spectrophotometry
Spectrophotometry is an analytical technique used to measure the intensity of light absorbed or transmitted by a substance at specific wavelengths, enabling quantification of analyte concentration in solution.
What is spectrophotometry?
Spectrophotometry is a fundamental analytical method in chemistry, biochemistry, and pharmaceutical sciences that quantifies how much light a sample absorbs or transmits across a range of wavelengths. It operates on the principle that different compounds absorb light at characteristic wavelengths, allowing identification and concentration determination. Instruments known as spectrophotometers measure absorbance (A) using Beer-Lambert Law: A = ε × c × l, where ε is the molar absorptivity, c is concentration, and l is path length. This technique is widely used in quality control, enzyme kinetics, nucleic acid and protein quantification (e.g., A260/A280 ratios), and drug stability testing.
How does spectrophotometry work?
A spectrophotometer emits a beam of light across a defined wavelength range (typically UV-Vis: 190–1100 nm). The light passes through a sample in a cuvette, and a detector measures the transmitted intensity. The instrument compares this to a reference (blank) to calculate absorbance. Modern instruments often use diffraction gratings or monochromators to isolate specific wavelengths. Data can be plotted as absorption spectra, revealing peak absorbance wavelengths unique to each compound. Applications include monitoring reaction progress, determining purity, and validating formulations.
What are the key applications in biotech and pharma?
In biotechnology and pharmaceutical development, spectrophotometry is essential for: verifying protein and nucleic acid concentrations (e.g., using Bradford or BCA assays); assessing drug stability under various conditions; measuring enzyme activity via substrate depletion; and ensuring compliance with regulatory standards such as USP, EP, and ISO. It is also used in environmental monitoring and food safety testing (e.g., FCC standards).
Related concepts
Commonly used alongside spectrophotometry are techniques such as HPLC, UV-Vis spectroscopy, NMR, and ELISA. Calibration with certified reference materials and adherence to ISO 17025 standards ensure data reliability.