UV-Visible Spectroscopy (UV-Vis)

UV-Visible Spectroscopy (UV-Vis)

Overview of UV-Vis Spectrometer

  • Components:
    • Light source
    • Chopper
    • Shutter
    • Monochromator
    • Sample compartment
    • Mirrors
    • Semitransparent mirror
    • Detector (PT)
    • Signal processor
    • Temperature control

Introduction

  • UV-Vis Spectroscopy uses light to measure chemical concentration.
  • Light is described as a photon because of its ability to carry energy.
  • E = hv
    • Where:
      • E is energy.
      • h is Planck's constant (6.626 x 10^{-34} J.s).
      • v is the frequency of light in Hz.

Electromagnetic Spectrum

  • Electromagnetic Spectrum ranges from Radio waves to Gamma rays.
  • Wavelengths range from 10^3 meters (Radio) to 10^{-12} meters (Gamma Ray).
  • Frequencies range from 10^4 Hz (Radio) to 10^{20} Hz (Gamma Ray).
  • Visible light is a small portion of the electromagnetic spectrum (.5 x 10^{-6}).

Electromagnetic Spectrum (Continued)

  • Radiant energy propagated as a transverse wave.
  • Vibrates perpendicular to the direction of propagation.

Wave Properties

  • Wavelength (\lambda): The distance of one complete cycle.
  • Frequency: The number of cycles passing a fixed point per unit time.

Wavenumber

  • Wavenumber is the reciprocal of the wavelength (cm⁻¹).
  • Relationship between wavelength and frequency:
    • \text{Wavelength (cm)} = \frac{\text{Velocity of light (}3 \times 10^{10} \text{ cm/s)}}{\text{Frequency (Hz or s}^{-1})} (corrected LaTeX)

UV-Vis Absorption

  • Electromagnetic radiation for the UV-Vis region ranges in wavelength from approximately 200 to 700 nm.
  • Ultraviolet (UV) range: 200 to 350 nm.
  • Visible range: 350 to 700 nm.

UV-Vis Absorption (Continued)

  • When white light passes through an object, certain wavelengths are absorbed, and the unabsorbed wavelengths are transmitted or reflected.

Measurement

  • The amount of light absorbed is measured as a function of wavelength (\lambda).
  • This provides qualitative and quantitative information about the sample.
  • \lambda_{max} is a characteristic value.
  • Absorbing compounds must be examined in diluted solution using solvents like water, ethanol, hexane, and cyclohexane.
  • Solvents with double or triple bonds or heavy atoms (e.g., S, Br, I) should be avoided.

Chromophore and Auxochrome

  • A chromophore refers to the absorbing groups of atoms in a molecule.
  • A molecule containing a chromophore is called a chromogen.
  • Absorption results from the transition from the ground state (lower energy) to excited states (higher energy).

Beer's Lambert Law

  • States the relationship between the absorbance of a solution and the concentration of the absorbing species.
  • Equation:
    • A = abc
    • A = \epsilon bc
    • Where:
      • A = absorbance (no unit)
      • a = absorptivity (L/g.cm)
      • \epsilon = molar absorptivity (L/mol.cm)
      • b = optical path length (cm)
      • c = concentration (g/L)

Attenuation

  • Describes how the amount of attenuation depends on the concentration of absorbing molecules and the path length over which absorption occurred.
  • Absorption may be presented as:
    • Transmittance (T = \frac{P}{P_0})
    • Absorbance (A = \log \frac{P_0}{P})
    • Where:
      • P_0 = power of incident radiation
      • P = power of transmitted radiation
      • c = concentration
      • b = path length

Transmittance and Absorbance

  • Transmittance is often expressed as a percentage (%T).
  • Absorbance is a logarithmic function of transmittance.

Absorbance Values

  • Absorbance is zero when transmittance is 100%, meaning no light is absorbed by the sample.
  • Absorbance will be infinite when transmittance is zero (all lights were absorbed by the sample).
  • When P = P_0, T = 1.0 or 100%, and A = 0.

Beer’s Lambert Law Implications

  • Referring to the Beer’s Lambert Law Equation [ A = \epsilon bc ] → the absorbance depends on the total quantity of the absorbing compound in the light path through the cuvette.

Standard Calibration Curves

  • A plot of absorbance vs. concentration will yield a straight line passing through the origin (0,0).
  • The linear relationship between concentration and absorbance is simple and straightforward.
  • The plot of %T vs. concentration will give an exponential-type graph (complicated relationship).

Limitations of Beer's Lambert Law

  • The Beer's Lambert Law is not obeyed at high concentrations.

Errors

  1. Gravimetric errors (e.g. mood, skills etc.)
  2. Incomplete spectral resolution (due to wrong slit width selection).
  3. Turbidity (must filter the sample to avoid cloudy samples).
  4. Aggregation at high concentration (the particles stick together).
  5. Contamination of cuvettes (must clean the cuvettes immediately after use).

Example of Calculation 1

  • A 7.50 x 10^{-5} M solution of potassium permanganate has a transmittance of 36.4% when measured in a 1.05 cm cell at a wavelength of 525 nm.
  • Calculate:
    • (a) absorbance of this solution
    • (b) molar absorptivity of KMnO4
  • Answer:
    • a) A = - \log T = - \log 0.364 = 0.439
    • b) Use: A = \epsilon b c
    • \epsilon = \frac{A}{bc} = \frac{0.439}{1.05 \text{cm} \times 7.50 \times 10^{-5} \text{mol/L}} = 5.57 \times 10^3 \text{L/mol.cm}

Example of Calculation 2

  • At 580 nm, the wavelength of maximum absorption of complex Fe(SCN)^{2+} has a molar absorptivity of 7.0 x 10^3 L/mol.cm.
  • Calculate the absorbance of a 2.5 x 10^{-5} M solution of the complex at 580 nm in a 1.0 cm cell.

Example of Calculation 3

  • Find the concentration of samples deer 1 & deer 2.
  • Plot a standard calibration curve.

Instrumentation: UV-Vis Spectrophotometer

  • Consists of:
    1. Light Source
    2. Wavelength selector (monochromator)
    3. Sample containers (quartz & glass)
    4. Detector
    5. Signal processor and readout

Single Beam Spectrometer

  • The simplest type of spectrometer employs a single source to supply radiation to the sample.
  • Well-suited for quantitative absorption measurement at single-wavelength type.
  • Simple, low cost, and ease of maintenance.
  • The sample & blank must be placed alternately in the light path (time-consuming)
  • For measurements at multiple wavelengths, the blank must be run at each wavelength.

Double Beam Spectrometer

  • Has two light paths: one for the sample & one for a blank or reference.
  • Measures both the sample and solvent.
  • The detection electronics manipulate the measurements to give the absorbance.

Double Beam Spectrometer Advantages

  • Automatic scanning & continuous recording of spectrum (absorbance versus wavelength).
  • The absorption in the sample is automatically corrected for the absorption occurring in the blank.
  • Automatic correction for changes of the source intensity & changes in the detector response with time or wavelength because the two beams are compared & measured at the same time.

Double Beam Spectrometer Disadvantages

  • What are the disadvantages of Double Beam Instrument?