Fluorescence spectrophotometry and UV-Vis spectrophotometry are related techniques, but they work differently and provide different types of information. Here’s a comparison:
1. Principle
- UV-Vis Spectrophotometry:
Measures the absorption of light by a sample at specific wavelengths.- Absorbance increases with concentration (Beer-Lambert law).
- Fluorescence Spectrophotometry:
Measures the light emitted by a sample after it absorbs light.- The sample is excited by a specific wavelength, then emits light at a longer wavelength.
2. Type of Measurement
- UV-Vis: Detects absorbed light.
- Fluorescence: Detects emitted light.
3. Sensitivity
- UV-Vis: Moderate sensitivity.
- Fluorescence: Much higher sensitivity; can detect very low concentrations (trace amounts).
4. Information Provided
- UV-Vis: Gives concentration and some structural clues.
- Fluorescence: Gives concentration and information about environment around the molecule, such as polarity or binding events.
5. Sample Requirements
- UV-Vis: Most compounds that absorb light can be measured.
- Fluorescence: Only fluorescent molecules or those that can be tagged with a fluorescent probe can be measured.
6. Wavelengths
- UV-Vis: Measures same wavelength for absorption.
- Fluorescence: Measures emission at a longer wavelength than excitation.
In short:
- UV-Vis spectrophotometry measures how much light a sample absorbs.
- Fluorescence spectrophotometry measures how much light a sample emits after absorbing light.
- Fluorescence is more sensitive but requires fluorescent molecules, while UV-Vis is more general.