Absorption Spectroscopy is an essential analytical technique used to measure how molecules absorb light at specific wavelengths. When a beam of electromagnetic radiation passes through a sample, part of the energy is absorbed as molecules transition from a lower energy state to a higher energy state. The amount of absorbed light provides valuable information about the structure, concentration, and functional groups of molecules. Absorption spectroscopy forms the foundation of UV-Visible spectrophotometry, widely used in pharmaceutical quality control, chemical analysis, research, and biochemical studies.
Absorption of Light
When light interacts with matter, electrons within atoms or molecules absorb energy and move to higher electronic energy levels. The amount of energy absorbed corresponds to specific wavelengths, making the absorption pattern a molecular signature. The basic principle involves measuring the decrease in intensity of light as it passes through a sample.
Principle and Measurement of Radiation
The principle of absorption spectroscopy is based on the Beer-Lambert law, which relates absorbance to concentration. A monochromatic beam of light is passed through the sample, and the transmitted light is measured. The instrument compares incident and transmitted light to calculate absorbance.
Absorbance is directly proportional to the analyte concentration, path length, and molar absorptivity. This simple and reliable relationship makes the technique ideal for quantitative analysis.
Electronic Transitions
Electronic transitions occur when electrons move between molecular orbitals. The main types of transitions include:
- σ → σ* transitions: Require high energy and occur in saturated compounds.
- n → σ* transitions: Involve non-bonding electrons and occur in molecules containing heteroatoms.
- π → π* transitions: Common in molecules with double bonds and aromatic systems.
- n → π* transitions: Occur in carbonyl-containing compounds and other chromophores.
1) Absorption in Species Containing π, σ, and n Electrons
Molecules containing π electrons (double bonds, aromatic rings) and non-bonding electrons (O, N, S) exhibit characteristic absorption in the UV-Visible range. These transitions produce strong absorption bands that help identify functional groups and molecular structures.
2) Absorption Involving d and f Electrons
Complex ions of transition metals show absorption due to d–d transitions, where electrons move between split d-orbitals. Lanthanides and actinides exhibit f–f transitions. These transitions give rise to colored complexes, widely used in metal ion analysis and pharmaceutical assays.
3) Charge-Transfer Absorption
Charge-transfer complexes form when an electron donor interacts with an electron acceptor. These complexes absorb strongly in the visible region, producing intense colors. Charge-transfer absorption is used in identifying trace metal ions and studying complex formation.
Chromophores
Chromophores are groups of atoms responsible for light absorption. Common chromophores include:
- C=C (alkenes)
- C=O (carbonyls)
- N=N (azo groups)
- Aromatic rings
A chromophore determines the wavelength at which a molecule absorbs and provides insights into its structural features.
Auxochromes
Auxochromes are functional groups (e.g., –OH, –NH2, –OCH3) that do not absorb strongly on their own but modify the absorption characteristics of chromophores. They increase intensity and may shift the absorption maximum, aiding in structural identification.
Factors Affecting Spectral Shift
Spectral shifts refer to changes in absorption maxima due to modifications in molecular environment. The main types include:
- Bathochromic shift (red shift): Absorption moves to longer wavelengths.
- Hypsochromic shift (blue shift): Absorption moves to shorter wavelengths.
- Hyperchromic effect: Increase in absorption intensity.
- Hypochromic effect: Decrease in absorption intensity.
These shifts provide clues about conjugation, solvent effects, and structural changes.
Solvent Effect on Absorption Spectra
Solvents influence the position and intensity of absorption bands. Polar solvents stabilize excited or ground states differently, causing shifts in absorption maxima. Protic solvents cause stronger interactions, while aprotic solvents lead to less pronounced effects. Solvent selection is therefore critical for reproducible spectral analysis.
Measurement of Transmittance and Absorbance
Transmittance (T) is the ratio of transmitted light to incident light. Absorbance (A) is calculated as:
A = –log(T)
Absorbance is more reliable for quantitative analysis because it follows a linear relationship with concentration.
Theory of Spectrophotometry
Spectrophotometry is based on comparing light intensity before and after passing through the sample. A monochromator isolates specific wavelengths, and the instrument measures absorbance at those wavelengths. This allows qualitative identification through spectral patterns and quantitative determination using calibration curves.
The Beer-Lambert Law
The Beer-Lambert law is the fundamental equation of absorption spectroscopy:
A = εbc
Where:
- A: Absorbance
- ε: Molar absorptivity
- b: Path length
- c: Concentration of analyte
The law enables accurate quantitative analysis. However, deviations occur at high concentrations, scattering media, or when chemical equilibria change.
Deviations to Beer’s Law
Deviations may be:
- Chemical deviations: Due to shifts in equilibrium.
- Instrumental deviations: Resulting from broadband light or stray radiation.
- Physical deviations: Caused by high concentration or scattering.
Instrumentation
Instrumentation in absorption spectroscopy includes photometers and spectrophotometers. The basic components of UV-Visible instruments include:
- Light source
- Monochromator
- Sample holder (cuvette)
- Detector
- Readout system
Photometer
A photometer measures the intensity of transmitted light using filters instead of monochromators. Although simpler and less expensive, it offers limited wavelength selectivity. It is suitable for routine colorimetric analysis.
Spectrophotometer
A spectrophotometer uses prisms or diffraction gratings to isolate narrow wavelength ranges. It offers superior resolution and accuracy. Modern double-beam spectrophotometers compensate for fluctuations in light source intensity and improve precision.
Components of UV-Visible Spectrophotometer
- Light Source: Deuterium lamp for UV region; tungsten lamp for visible region.
- Monochromator: Isolates specific wavelengths.
- Cuvette: Usually made of quartz or glass.
- Detector: Converts light into electrical signals (e.g., photodiodes, PMTs).
- Readout System: Displays absorbance or transmittance.
Applications of UV-Visible Spectroscopy
- Quantitative analysis of drugs and pharmaceuticals
- Purity testing and impurity profiling
- Identification of chromophores and functional groups
- Study of reaction kinetics
- Analysis of transition metal complexes
- Molecular weight estimation of polymers
- Environmental and water quality monitoring
Detailed Notes:
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