Ultraviolet–Visible (UV–Vis) spectroscopy is an analytical technique used to measure the absorption of electromagnetic radiation by substances in the ultraviolet (200−400 nm) and visible (400−800 nm) regions of the spectrum.
- In this spectral range, absorption of light leads to electronic transitions in atoms and molecules. The common transitions include:
- σ → σ* (high energy, usually <200 nm)
- π → π* (commonly observed in unsaturated systems)
- n → π* (seen in molecules with lone pairs, e.g., carbonyls)
Principle
Uv-Vis spectroscopy is based on the absorption of electromagnetic radiation by molecules.
- Molecules containing bonding (σ, π) and non-bonding (n) electrons absorb energy.
- This causes excitation from: Lower-energy orbitals → higher-energy antibonding orbitals (σ*, π*).
- The amount of light absorbed is related to concentration by the Beer–Lambert Law:
\mathrm{A=\varepsilon bC}
Where A is the absorbance (unitless), ε is the molar extinction coefficient (L.mol-1.cm-1), C is the concentration (mol.L-1), and b is the path length (cm).
Working
The basic layout of a double-beam UV–Visible spectrophotometer consists of a radiation source, wavelength selector (monochromator), beam splitter, sample and reference cells, detector, and signal processing system.
- Radiation Source: Radiation from a suitable light source (typically a deuterium lamp for UV and a tungsten lamp for the visible region) is directed through a system of mirrors.
- These mirrors guide the light into the wavelength selection unit.
- Wavelength Selection: The light enters a monochromator, whose function is to isolate a narrow band of wavelengths from the polychromatic radiation.
- This is achieved using dispersing elements such as prisms or diffraction gratings.
- The selected wavelength exits through an exit slit, ensuring nearly monochromatic radiation.
- Beam Splitting: The monochromatic radiation then falls on a rotating sector mirror (or beam splitter).
- This device alternately divides the incoming beam into two separate beams:
- One beam passes through the reference (blank) cell
- The other passes through the sample cell
- This device alternately divides the incoming beam into two separate beams:
- Interaction with Sample and Reference: The reference cell contains the solvent or blank, while the sample cell contains the analyte solution.
- As light passes through these cells, part of it is absorbed depending on the sample’s properties.
- Detection of Radiation: The transmitted light from both paths is directed onto a detector (commonly a photodiode or photomultiplier tube).
- The detector converts the light energy into an electrical signal.
- Signal Processing: The detector output is sent to an amplifier, which enhances the signal.
- The system continuously compares the intensity of light passing through the sample and reference.
- Output and Recording: The instrument calculates transmittance (T) or absorbance (A) of the sample relative to the reference.
- The result is recorded as a function of wavelength, producing a UV–Vis spectrum.
Instrumentation:
A typical UV–Visible spectrophotometer consists of the following major components: light source, wavelength selector, sample holder, detector and signal processing System.
Light Source:
The light source provides radiation in the ultraviolet (UV) and visible (Vis) regions. An ideal source should be stable, intense, and continuous over the required wavelength range.
Hydrogen Discharge Lamp (UV Source)::
Used as a radiation source for the ultraviolet (UV) region in UV–Visible spectroscopy.
- Construction: Hydrogen gas is enclosed under high pressure inside the lamp.
- Equipped with a cathode (W) and an anode (Ni) to allow electric discharge through the gas
- Working: When an electric discharge passes through the gas, hydrogen molecules become electronically excited and emit ultraviolet radiation.
- Due to frequent molecular collisions at high pressure, a continuous spectrum is produced rather than discrete lines.
- Wavelength range: approximately 150–350 nm.
- Features: Stable, robust, and widely used in older instruments.
Deuterium Lamp (UV Source)::
A modified version of the hydrogen discharge lamp, using deuterium gas (heavy hydrogen) instead of hydrogen. It is the most widely used UV radiation source in modern UV–Visible spectrophotometers.
- Construction: Deuterium gas is enclosed under high pressure inside the quartz lamp. Quartz is used because it transmits UV radiation effectively.
- Equipped with a cathode (W) and an anode (Ni) to allow electric discharge through the gas
- Working: When an electric discharge passes through deuterium gas. Deuterium molecules become electronically excited and emit ultraviolet radiation on relaxation.
- Due to frequent molecular collisions at high pressure, a continuous UV spectrum is produced.
- Wavelength range: Approximately 160–375 nm
- Features: Preferred where high intensity is required, more expensive than hydrogen lamps, provides a stable and consistent UV output, has higher emission intensity (about 3–5 times that of a hydrogen lamp of similar design), Limited lifetime compared to some other light sources.
Tungsten–Halogen Lamp (Visible Source)::
Used as the primary source for the visible region in UV-vis spectroscopy.
- Construction: It consists of a tungsten filament enclosed in a quartz glass filled with a small amount of halogen gas such as iodine or bromine.
- Working: When electric current passes through the filament, it is heated to a high temperature (~2500–3000 K).
Source\;\rightarrow\;Monochromator\;\rightarrow\;Sample\;\rightarrow\;Detector