UV Visible Spectroscopy 3

Background on Spectroscopy

  • Spectroscopy: Study of electromagnetic radiation and its interaction with substances, providing qualitative and quantitative information.

    • Light: Form of EMR (electromagnetic radiation) that carries energy.

    • Wavelength (λ): Linear distance from crest to crest of a wave.

    • Frequency (ν): Number of waves per second (measured in CPS or Hz).

    • Speed of Light (c): Formula: c = λ × ν (293,000 km/s).

    • Energy (E): Formula: E = hν = hc/λ; where h = Planck's constant (3.63 x 10^-27 erg.s).

Electromagnetic Radiation Spectrum

  • Energy transitions associated with various electromagnetic spectrum regions:

    • Nuclear spin (NMR)

    • Electronic spin (electron spin resonance)

    • Radiofrequency

    • Microwave (Rotational)

    • Infrared (IR) (Vibrational)

    • Ultraviolet/Visible (UV/Vis) (Electronic)

    • X-rays (Bond breaking)

UV/Visible Light Spectrum

  • UV Spectrum: 200 - 400 nm

  • Visible Light Spectrum: 400 - 900 nm

  • Molecular Absorption of UV-Vis Light:

    • Electronic transitions of molecular orbitals:

      • σ: Bonding electrons of sigma covalent bonds (lowest energy).

      • π: Bonding electrons in pi covalent bonds (higher energy than σ).

      • Non-bonding (n): Electrons that don’t participate in bonding (highest energy level).

Interaction of Light with Molecules

  • Excitation and Relaxation:

    • ΔE (Transitional Energy) = absorbed light.

    • ν = frequency of EMR absorbed.

    • ΔE determines the light wavelength absorbed.

    • Equation: ΔE = Es - Eg = hν = hc/λ.

Requirements for Absorption

  • Molecules must have conjugated double bonds to absorb in the range of 200-900 nm.

  • Chromophore: Atom group that interacts with light and imparts color. More conjugated = longer wavelength absorption.

Factors Affecting Light Absorption

  • Absorbance Spectra: Graphical representation of absorbance at different wavelengths.

  • Factors affecting absorption include:

    • Concentration of the absorbing solution.

    • Path length of the sample cell.

    • Nature of the absorbing substance.

Laws of Spectrophotometry

  • Transmittance (T): Ratio of light intensity passing through a sample.

    • Formula: T = I/I0 (percentage transmittance = T × 100).

  • Bouguer-Lambert Law: Describes exponential decay of intensity with path length and concentration:

    • I = I0 e^(-k × l) for path length.

    • I = I0 e^(-k' × c) for concentration.

Beer-Lambert Law

  • Defines relationship between absorbance (A), concentration (c), and path length (l):

    • A = ε × c × l

    • ε = molar absorption coefficient.

Molar Absorption Coefficient

  • If l is in cm and c in mol/L, ε has units L mol⁻¹ cm⁻¹.

  • Useful for comparing absorption abilities of different substances on a molecular basis.

Specific Absorbance

  • Defined for solutions in % m/v (g/100 mL). Denoted as A(1%, 1 cm).

Experimental Measurement of UV-Vis

  • Components of a UV-Vis spectrophotometer:

    • Light Source: Tungsten (Vis) & Deuterium (UV).

    • Monochromator: Filter/Prism/Grating.

    • Sample compartment: Cuvette.

    • Detector: Photocell/Photomultiplier Tube.

Quantitative Analysis Methods

  1. Calibration Curve: Use multiple standard solutions to measure absorbance and construct a curve for determining sample concentration.

  2. Beer-Lambert Law: Calculate concentration from absorbance and known absorption coefficient.