Spectroscopic Techniques

SPECTROSCOPY - Introduction

• Definition: The study of the interaction of electromagnetic radiation with molecules in a sample for identification, quantification, and characterization.
• Types of Interaction:
  • Absorption
  • Emission
  • Scattering
• Instruments Used: Specialized electronic instruments for studying spectroscopy are known as spectrophotometers.

SPECTROSCOPY - Electromagnetic Radiation

• Nature of Electromagnetic Radiation: A form of radiation where electric and magnetic fields vary simultaneously.
• Characterization:
  • Energy
  • Intensity
  • Frequency
  • Wavelength
• Examples:
  • White light from the sun or electric bulbs (visible light)
  • Infrared rays
  • Ultraviolet rays
  • X-rays
• Energy-Wavelength Relationship:
  • The energy of electromagnetic radiation is inversely proportional to the wavelength:
  • Long wavelength → Low energy
  • Short wavelength → High energy
• Equations:
  • $E = h<br />\nu$
  • where $E$ = energy, $h$ = Planck's constant, $<br />\nu$ = frequency
  • $<br />\nu = \frac{c}{ ext{λ}}$
  • Thus, $E = \frac{hc}{ ext{λ}}$

SPECTROSCOPY - Electromagnetic Spectrum

• Definition: The continuous range of all types of electromagnetic radiation arranged according to wavelength, frequency, or energy.
• Range: From long wavelength (radio waves) to short wavelength (gamma rays).

SPECTROSCOPY - Electromagnetic Spectrum Regions

SPECTROSCOPY - Classification of Spectroscopy

1. Absorption Spectroscopy

• Definition: When a molecule or atom interacts with electromagnetic radiation.
• Absorption Process:
  • Atoms/molecules absorb energy, promoting them to higher energy levels (excitation).
  • The absorbed energy is used for quantification and identification.
• Examples: Atomic absorption spectroscopy, UV-visible spectroscopy, infrared spectroscopy (IR).

2. Emission Spectroscopy

• Process: Atoms/molecules absorb energy, become excited, and then emit light upon returning to lower energy states.
• Characteristics: The emitted light has characteristic wavelengths allowing quantification and identification.
• Examples: Atomic emission spectroscopy, Fluorescence spectroscopy.

3. Scattering Spectroscopy

• Definition: Atoms/molecules redirect electromagnetic radiation causing scattering.
• Characteristics: Scattering can cause polarization and changes in spectral features.
• Examples: Rayleigh scattering, Raman spectroscopy.

SPECTROSCOPY - Types of Spectroscopy

A. Molecular Spectroscopy

• Definition: The study of interaction between molecules and electromagnetic radiation.
• Types:
  • Ultraviolet-visible (UV-vis) spectroscopy
  • Infrared (IR) spectroscopy
  • Raman spectroscopy
  • Fluorescence spectroscopy
  • Nuclear Magnetic Resonance (NMR) spectroscopy
  • Mass Spectrometry (MS)

B. Atomic Spectroscopy

• Definition: The study of interaction between atoms and electromagnetic radiation.
• Types:
  • Atomic Emission spectroscopy
  • Atomic Absorption spectroscopy

ULTRAVIOLET - VISIBLE (UV-vis) SPECTROSCOPY – Introduction

• Definition: Analytical technique measuring the absorption of UV and visible light by molecules.
• Use: Useful for qualitative and quantitative analysis of compounds.

ULTRAVIOLET - VISIBLE (UV-vis) SPECTROSCOPY – Principle

• Mechanism: When UV (200-400 nm) or visible light (400-800 nm) passes through a molecule, certain wavelengths are absorbed, causing electronic excitation of electrons.
• Quantitative Analysis: The amount of light absorbed is proportional to the concentration of the absorbing species according to Beer-Lambert Law.

ULTRAVIOLET - VISIBLE (UV-vis) SPECTROSCOPY – Types of Electronic Transitions

• Types of Transitions:
  • ext{σ}
    ightarrow ext{σ}^*
  • n
    ightarrow ext{σ}^*
  • n
    ightarrow ext{π}^*
  • ext{π}
    ightarrow ext{π}^*
• Description: Each type of transition depends on the nature of electrons involved and molecular structure.

Transitions

1. ext{σ} ightarrow ext{σ}^*

   • Requires high energy and occurs in saturated compounds.
   • Example: Alkanes.

2. n ightarrow ext{σ}^*

   • Involves non-bonding (n) electrons from heteroatoms.
   • Examples include alcohols, amines, and alkyl halides.

3. n ightarrow ext{π}^*

   • Involves promotion of an electron from a non-bonding orbital to a π antibonding orbital.
   • Example: Carbonyl compounds and nitro compounds.

4. ext{π} ightarrow ext{π}^*

   • Occurs in unsaturated systems like alkenes and aromatic compounds, requiring lesser energy than sigma transitions.

ULTRAVIOLET - VISIBLE (UV-vis) SPECTROSCOPY – Instrumentation

• Components of a UV-vis Spectrophotometer:
  1. Radiation source
  2. Monochromator
  3. Sample holder (cuvette)
  4. Detector
  5. Readout system

1. Radiation Source

• Function: Produces continuous radiation over wavelength range.
• Types:
  • Deuterium (D₂) Lamp: UV Region (190 – 400 nm)
  • Tungsten Lamp: Visible Region (350 – 800 nm)
  • Xenon Arc Lamp: Suitable for both UV and visible ranges.

2. Monochromator

• Function: Isolates specific wavelength from polychromatic light.
  • Types include prism monochromators and diffraction grating monochromators.

3. Sample Holder (Cuvette)

• Specifications: Interacts with monochromatic light and typically has a pathlength of 1 cm.
  • Types:
  • Quartz: Suitable for UV and visible range
  • Plastic: Suitable for visible range
  • Glass: Suitable for visible range only

4. Detector

• Function: Measures the intensity before and after passing through the sample to identify and quantify analytes.

5. Readout System

• Function: Displays analytical information such as absorbance, transmittance, and spectra.
  • Features of Spectrum: Characterized by $λ_{max}$ (wavelength of maximum absorbance).

Factors Affecting Wavelength of Absorption in UV-vis Spectroscopy

1. Nature of Chromophore: Responsible for light absorption; energy gaps affect wavelength absorption (bathochromic/red shift or hypsochromic/blue shift).
2. Extent of Conjugation: More conjugation implies greater delocalization of $ext{π}$-electrons leading to lower energy transitions.
3. Substituents (Auxochromes): Groups modifying absorption characteristics; electron-donating groups increase electron density affecting absorption wavelength.
4. Solvent Effects: Stabilization effects of solvents on ground state or excited state impact absorption wavelength.
5. pH of the Medium: Different ionic forms absorb differently depending on pH, thus affecting absorption characteristics.

Analysis and Applications of UV-vis Spectroscopy

• Qualitative and Quantitative Analysis: Using absorbance at specific $λ_{max}$ and constructing calibration curves.
• Applications: Used in pharmaceutical analysis, macromolecular analysis, and in detectors for HPLC techniques.

Limitations of Beer-Lambert Law

1. Fundamental limitations: Interaction of solutes altering behavior at higher concentrations (>0.01 M). Light scattering effects.
2. Chemical limitations: Changes in chemical properties altering concentration of absorbing species leads to measurement errors (aggregation/dissociation, re-emission of absorbed light).
3. Instrumental limitations: Assumes monochromatic light but many instruments utilize bands, introducing inaccuracies due to stray light and calibration errors.