Absorption spectroscopy measures how materials absorb light at different wavelengths. It's a powerful tool for identifying and quantifying substances in various fields. The Beer-Lambert law is key, linking light absorption to sample concentration and path length.
This technique has wide-ranging applications, from environmental monitoring to pharmaceutical analysis. Factors like concentration, path length, and wavelength affect absorption spectra, influencing the accuracy and sensitivity of measurements. It's a versatile, non-destructive method for chemical analysis.
Principles and Instrumentation
Principles of absorption spectroscopy
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Measures absorption of electromagnetic radiation by a sample as a function of wavelength or frequency
Incident light attenuated by the sample, transmitted light measured
Absorption occurs when energy of incident photons matches energy difference between ground state and excited state of sample molecules
Allows identification and quantification of chemical species based on their characteristic absorption spectra (UV-Vis, IR)
Non-destructive technique, sample can be recovered after analysis
Sensitivity and selectivity depend on the choice of wavelength and sample preparation
Beer-Lambert law in quantitative analysis
Relates attenuation of light to properties of material through which light is traveling
Absorbance (A A A ) directly proportional to concentration (c c c ) of absorbing species and path length (l l l ) of sample: A = ε l c A = \varepsilon lc A = εl c
ε \varepsilon ε is molar attenuation coefficient, constant characteristic of absorbing species at particular wavelength
Prepare series of standard solutions with known concentrations of analyte
Measure absorbance of standard solutions and create calibration curve (absorbance vs. concentration)
Measure absorbance of unknown sample and determine concentration using calibration curve
Allows accurate quantification of analytes in various matrices (solutions, gases)
Factors affecting absorption spectra
Concentration
Higher concentrations lead to increased absorbance, more molecules available to absorb light
Deviations from linearity may occur at high concentrations due to aggregation or saturation effects
Path length
Longer path lengths result in increased absorbance, light interacts with more sample molecules
Doubling path length doubles absorbance, as per Beer-Lambert law
Choose appropriate path length to ensure absorbance falls within linear range of instrument (0.1-1.0)
Wavelength of incident light
Absorption occurs when energy of incident photons matches energy difference between molecular states
Different compounds absorb light at different wavelengths, leading to characteristic absorption spectra
Wavelength of maximum absorption (λ m a x \lambda_{max} λ ma x ) often used for quantitative analysis
Choose wavelength with high molar attenuation coefficient for increased sensitivity
Applications of absorption spectroscopy
Environmental monitoring
Determine concentration of pollutants in water or air samples (heavy metals, organic contaminants)
Monitor water quality in rivers, lakes, and oceans
Assess air quality in urban and industrial areas
Pharmaceutical analysis
Verify identity and purity of drug compounds
Quantify active ingredients and impurities in formulations
Ensure compliance with regulatory standards (USP, EP)
Biochemical assays
Quantify concentration of biomolecules in biological samples (proteins, nucleic acids)
Monitor enzymatic reactions and kinetics
Study protein-ligand interactions and binding affinity
Material science
Characterize optical properties of materials (semiconductors, polymers)
Investigate electronic transitions and band structure
Assess purity and composition of materials
Food and beverage industry
Determine nutritional content of food products (vitamins, minerals)
Monitor fermentation processes in beer and wine production
Detect adulteration and contamination in food samples