Spectroscopy– studies the interaction between electromagnetic radiation (light) and matter.
It broadly falls under three categories–
- Absorption spectroscopy- light is absorbed by a sample
- Emmision spectroscopy- light is released by a sampled
- Scattering- light is scattered by a sample
What it can do?
Detect chemical composition, find molecular structure, and even tell us the physical properties of a sample.
Types of spectroscopy- atomic, IR, molecular, UV-vis, NMR, Xray
Principle of spectroscopy is governed by the Beer-Lambert’s law
Beer lambert’s law
Statement- The absorbance (A) of light passing through a sample is directly proportional to the concentration of the sample (c) and path length (l).
ε is molar absorbivity or extinction coefficient. It measures how strongly a substance absorbs light at a given wavelength per molar concentration. So, higher concentration or high path length results in higher absorbance.
Transmittance (T)
It measures the amount of light that passes through the sample. It ranges from 0-100%. It represents how much radiation passes through the sample without being absorbed or reflected.
P0= fraction of incident light
Percentage Transmittance= %T= [P/P0] * 100
Relationship between %T or T and A
It is used for quantifying concentration of substances.
Light sources
Key light sources include-
- Deuterium lamps- 160-375 nm for UV light
- Tungsten/ halogen lamps- 320-2300 nm for visible light
- Xenon arc lamps- 190-1100 nm for high intensity lights
- Laser/ LED lamps- for monochromatic narrow wavelength applications
Monochromator– it is an optical device used in spectroscopy. It can isolate narrow band of wavelength (monochromatic light) from a polychromatic source. It utilizes a diffracting prism with entrance and exit slits to select specific wavelengths. The entrance slit accepts the input light and the collimating mirrors renders the light rays parallel. The prism then splits the light into its components (spectrum). The focusing mirror  focusses the dispersed spectrum onto the exit slit allowing a narrow wavelength band to pass to the detector.
Types of detectors used in spectroscopy
A detector measures radiant energy by converting photons or thermal energy into digital signals.
- Photon/ Quantum detectors- suitable for near IR or UV-Vis spectrum
- Photomultiplier tubes- best for low intensity measurements
- Silicone photodiodes- they convert light into current. Best for multiple wavelengths
- Photoemmisive cells- photons strike a photoemissive cathode and release electrons
- Charge-Coupled devices- used in raman spectroscopy. Highly sensitive and capable of image acquisition.
- Thermal detectors
They absorb radiation caused by a change in temperature. This signal is converted to a digital reading. These types of detectors are mostly used in IR spectroscopy.- Thermocouples- voltage is proportional to temperature difference between two junctions.
- Thermistors- measure change in resistnace caused by temperature changes
- Pyroelectric detectors- measure change in polarization of a material
Other detectors
- Faraday cup- measure ion currents. Used in mass spectrometers
- Flame ionization detectors- measure ions produced by burning samples in a flame. Used in GC- spectroscopy
Types of spectroscopy- working principle and applications
| Type | Wavelength | What it measures | Principle | Quantified using | Results are shown in the form of | Applications |
| Visible spectroscopy | 400 to 800 nm | absorption of light by molecules | Upon absorption of light, valence electrons get excited from ground state to higher energy orbitals. This transition corresponds to specific absorbed wavelengths. This way, we can identify key functional groups present in the sample. | Beer Lambert’s Law | Spectral data, a plot of light absorbance against wavelength | Pharmaceuticals (drug identification), food & beverage (quality control), foresnsics (trace evidence analysis), Lifesciences (Quantification of biomolecules) |
| UV- visible spectroscopy | 190-800 nm | absorption of light by molecules | Upon absorption of light, valence electrons get excited from ground state to higher energy orbitals. This transition corresponds to specific absorbed wavelengths. This way, we can identify key functional groups present in the sample. | Beer Lambert’s Law | absorption (obtained when electrons excite to higher energy levels) and emmision spectra (obtained when electrons come back to ground state) | Molecular biology (DNA and RNA analysis), pharmaceuticals (drug identification), bacterial culture (optical density- 600 nm is ideal) |
| IR spectroscopy | 2500 to 16000 nm | Vibrational transitions in a molecule | Covalent bond acts like a spring holding two atoms together. They vibrate two ways- 1. stretching: changing the bond length between two atoms or by 2. bending: changing the bond angle between the bonds (scissoring, rocking, wagging, twisting) If the frequency of the incedent IR light matches the vibration frequency of the bond, it absorbs energy and vibrates faster. Fingerprint Region: is unique to each molecule, allowing identification by comparing the spectrum to known standards. | Beer-lambert’s law | a spectrum showing absorbance vs transmittance | structure identification, forensic science, pharmaceuticals, and environmental analysis. |
| Raman spectroscopy | 244 to 1064 nm | inelastic scattering of light (raman effect) | When monochromatic laser light hits the sample, most light scatters elastically (Rayleigh scattering). A tiny fraction scatters inelastically exchanging energy with molecular vibrations and that is the raman effect. | Calibration curve | spectrum showing intensity vs wave number | Pharmaceuticals, cosmetics, Material science and nanotechnology, Biomedical and life sciences, Foresnsics |
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