Please enable JavaScript.
Coggle requires JavaScript to display documents.
Spectrophotometer, 6604071610194 น.ส.มันฑะนา ด้วงแตง - Coggle Diagram
Spectrophotometer
Theory and Principles of UV-1280
Light and the Electromagnetic Spectrum
• An electromagnetic radiation consisting of 2 components: Electric and magnetic field
• Both fields intersect each other at right angle
• Characterize by wavelength (l) - Distance between 2 adjacent wave crests
Electronic Transitions, Color, and Absorption
• Molecules absorbing electromagnetic radiation results in
– Electronic transition
– Vibrational motion
– Rotational motion
• Increase in molecule’s total energy
• When white light falls upon a sample, reflection and absorption occurl
• If only a portion of the light is absorbed, the color of the sample is determined by the reflected light
Beer-Lambert’s Law
A = ϵcl
ϵ : Molar absorptivity
l : Pathlength
c : Analyte concentration
Calibration Curve
Limitations in UV-Vis
Increase in analyte concentration
Chemical deviation due to physico-chemical interaction
Stray light
Absorption and Scattering by Cell Wall
Types of Cuvettes
• Quartz or Fused silica
• Silicate glass
• Plastics
UV-1280 Instrumentations
Spectrophotometer Structure
A spectrophotometer consists of four components
1) light source
The UV-Vis instrument has 2 light sources : Deuterium (D2) lamp and Tungsten
halogen (WI) lamp
Deuterium (D2) lamp
• Source for UV region
• Wavelength range : 190 – 350 nm
Tungsten halogen (WI) lamp
• Source for Visible region
• Wavelength range* : 350 – 900 nm
2) monochromator
The slit width will affect the intensity of the light irradiating the sample and resolution.
• Greater light intensity produces data that is
less influenced by noise.
• In general, it is a good practice to narrow slit with no more than it is necessary in order to better resolve the spectrum.
3) sample compartment
4) detector
Silicon Photodiode
detector (P.D.)
Photomultiplier Tube
(PMT)
Spectrophotometer Types
• Single-Beam Spectrophotometer
Blank measurement : S0 energy recorded
Sample measurement : S1 energy recorded
• Double-Beam Spectrophotometer
Blank measurement : Ratio S0/R0 recorded
Sample measurement: Ratio S1/R1 recorded
UV-1280 Application : Kinetic
Kinetic Mode : Local Control
Photometric
Spectrum
Quantitation
Kinatics
Time Scan
Multi-Component
Bio-Method
Utilities
6604071610194 น.ส.มันฑะนา ด้วงแตง