GE-109
UV-Visible Spectrophotometry
Name: Ritesh Pahari
Group Number: 8
Roll No: 2024EEB1257
Objective:
To determine the concentration of a given contaminant in a sample using UV-Visible
spectrophotometry.
Working Principle:
UV-Vis spectrophotometry works on the basis of Beer-Lambert Law, which states that the
absorbance (A) of light by a solution is directly proportional to the concentration (c) of the absorbing
species and the path length (l) of the sample:
Where:
• = Absorbance
• = Intensity of incident light
• = Intensity of transmitted light
• = Molar absorptivity
• = Concentration
• = Path length
When UV or visible light passes through a sample, molecules absorb energy and transition from a
lower to a higher electronic energy level. The extent of absorption helps determine the sample
concentration.
Brief History / Evolution:
• 17th Century: The origin of spectrophotometry traces back to Isaac Newton, who
demonstrated the splitting of visible light into a spectrum using a prism.
• Early 20th Century: Scientists identified the UV region as a distinct part of the
electromagnetic spectrum. In 1913, Bohr's atomic model clarified the concept of electronic
transitions.
• Technological Development (1930s–1950s): First commercial UV-Vis spectrophotometers
were introduced. Arnold Beckman played a key role by developing instruments capable of
precise measurements.
• Modern Era: Advancements in optics, electronics, and computer integration led to the
development of double beam UV-Vis spectrophotometers, micro-volume systems, and
software-based analysis tools.
Components:
1. Lamp: Provides the initial beam of light with a range of 190–1100 nm.
o UV region (200–400 nm): Deuterium lamp
o Visible region (400–800 nm): Tungsten lamp
2. Monochromator: Selects a specific wavelength of light using prisms or diffraction gratings to
isolate the desired wavelength.
3. Sample Holder (Cuvette): Holds the sample. Quartz cuvettes are used for UV region due to
their transparency. Typical path length is 1 cm.
4. Detector: Measures the transmitted light intensity and converts it to an electrical signal.
5. Display/Data System: Displays absorbance or transmittance values. Includes a computer for
plotting spectra or calculating concentration. A plot of absorbance vs. wavelength shows
peaks that indicate higher concentration.
Detailed Working with Schematics: The light from the lamp passes through a monochromator to
select the required wavelength. The beam then enters the cuvette containing the sample. Depending
on the sample’s absorbance, some light is absorbed while the rest is transmitted. The detector
captures the transmitted light, and the signal is processed to display absorbance data.
(Schematic can be inserted here with the flow: Lamp → Monochromator → Cuvette → Detector →
Display)
Applications:
1. Pharmaceutical Industry:
o Drug identification
o Purity and concentration analysis of active pharmaceutical ingredients (APIs)
2. Biochemistry & Molecular Biology:
o Protein contamination analysis (280 nm)
o RNA/DNA quantification (260 nm)
3. Environmental Analysis:
o Detection of pollutants like nitrates, phosphates, heavy metals, and organic
compounds in water
4. Chemical Industry:
o Determining concentration of unknown samples
o Analyzing reaction rates and equilibrium constants
Advantages:
• Can detect very small quantities
• Sample can be recovered post-analysis
• Low cost per test; easy to operate
• Easy data processing and plotting
Limitations:
• Cannot detect compounds that do not absorb UV light
• Turbid/colored samples may scatter light and affect readings
• Requires quartz cuvettes for UV range, which are costly and fragile
• Needs calibration for accurate quantification