Notes on UV Visible Spectroscopy

Introduction

• Overview of UV Visible Spectroscopy
• Request from subscribers after previous videos on IR and NMR spectroscopy.
• Importance in chemistry labs and life sciences.

Purpose of UV Visible Spectroscopy

• Determining Concentration:
  • Used to find concentrations of different substances in a solution.
  • Can measure concentration of cells in microbiology.
• Data Quality:
  • Useful for concentration analysis, but less reliable for determining specific chemical groups due to similar data for different groups.

Colorimetry

• Related to UV Visible Spectroscopy:
  • Involves the measurement of colored substances to identify concentrations.
  • Extensively used in enzymatic reactions for enzyme activity and kinetics studies.

Mechanism of UV Visible Spectroscopy

Instrument Setup

1. Light Source:
   • Uses UV or visible light as an energy source.
   • Electromagnetic spectrum ranges from gamma rays to microwaves.
2. Monochromator:
   • Separates light into a single beam of a specific wavelength.
3. Beam Separator:
   • Guides the light into two chambers: control and sample.
4. Detector:
   • Measures the intensity of light before and after passing through the sample.

Light and Molecule Interaction

• Absorption Process:
  • Molecules absorb light energy, exciting electrons from ground state to higher energy state.
  • Absorption leads to decrease in light intensity, which is measured.
• Intensity Measurement:
  • Intensity of incident light (I0) vs. intensity of transmitted light (I).
  • Transmittance (T) and Absorbance (A):
    • T = I/I0
    • A = log(I0/I)
• Lambda Max (λmax):
  • Specific wavelength where maximum absorbance occurs for certain chemical groups.

Data Interpretation

• Graphical Representation:
  • Typically plots absorbance against concentration.
  • Linear relationships are easier to extrapolate for unknown concentrations.
• Beer-Lambert Law:
  • A = εCL
    • A: Absorbance
    • ε: Molar absorption coefficient
    • C: Concentration
    • L: Path length of the cuvette.

Molecular Mechanism

• Molecules have specific energy states (ground state, non-bonding, bonding states).
• Absorption of light leads to electron transitions (e.g., from sigma to sigma* or pi to pi*).
• Excitation Mechanism:
  • Energy absorbed corresponds to specific wavelengths, which vary per molecule.
• Limitations:
  • UV Visible Spectroscopy is primarily for concentration analysis, not identifying specific chemical bonds.

Conclusion

• UV Visible Spectroscopy is essential for analyzing concentrations in various solutions but has limitations in identifying specific chemical groups or bonds compared to other techniques (e.g., NMR).
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