Spectroscopy Notes

Spectroscopy and Spectrometry

• Spectroscopy Definition: The study of the interaction between radiation/energy/light (characterized by wavelength or frequency) and matter.
• Spectrometry Definition: A technique used to quantitatively measure substances based on spectroscopic principles.
• Spectrophotometry Definition: A specific spectrometric technique used to measure the quantity of substances based on UV-Vis and infrared spectroscopy.

Electromagnetic Radiation

• Electromagnetic radiation is energy emitted through space at high velocity. Examples include gamma rays, X-rays, UV, visible light, infrared, microwaves, and radio waves.
• It exhibits dualistic properties: wave-like (with mutually perpendicular magnetic components) and particle-like (photons carrying quanta of energy).
• Planck's Equation: $E = h \cdot v$ where:
  • $E$ = energy per photon
  • $h$ = Planck's constant ($6.63 x 10^{-34} J \cdot s$)
  • $v$ = frequency of light
• Also expressed as $E = h \cdot \frac{c}{\lambda}$, where $c$ is the speed of light ($3 x 10^8 m \cdot s^{-1}$) and $\\lambda$ is the wavelength.

Types of Spectroscopy

• Based on the state of matter: atomic spectroscopy (for atoms) and molecular spectroscopy (for molecules).
• Matter contains energy, which is the potential to do work.
• Types of Spectroscopy Based on Energy States:
  • UV Spectroscopy: Electronic energy states of conjugated molecules, carbonyl groups, nitro groups.
  • Infrared (IR) Spectroscopy: Vibrational energy states of functional groups and bond structures.
  • NMR Spectroscopy: Nuclear spin states for the number, type, and relative position of protons.
  • Mass Spectroscopy: High-energy electron bombardment to determine molecular weight, presence of nitrogen, and halogens.

Spectroscopic Techniques

• Absorption: Measures the reduction in light intensity after passing through a substance.
• Emission: Measures the electromagnetic spectrum emitted by a substance.
• Fluorescence: Based on the absorption of energy by a substance.
• Transmittance: The fraction of light that passes through a solution. It is inversely proportional to absorbance.

Lambert-Beer Law

• Equation: $A = abc$ (Absorbance = absorptivity x path length x concentration)
  • Also expressed as $A = -log T$ or $A = log(1/T)$, where A=absorbance and T=Transmittance.
• Where:
  • $A$ = Absorbance
  • $T$ = Transmittance
  • $a$ = Absorptivity (L mol-1cm-1)
  • $b$ = Path length/cuvette thickness (cm)
  • $c$ = Concentration of the solution (mol L-1)
• If monochromatic light with intensity $I<em>o$ passes perpendicularly through a block of thickness $b$ containing absorbing particles, the intensity decreases to $I</em>t$.

Conditions for Lambert-Beer Law Validity

• Low concentration
• Stable analyte
• Monochromatic light
• Clear solution

Spectrophotometer Components

• Light Source (Lamp): Emits all colors of light (white light).
• Monochromator: Selects a specific wavelength to transmit through the sample.
• Detector: Detects the wavelengths of light that have passed through the sample.
• Amplifier: Amplifies the signal for easier reading against background noise.

Light Sources

• UV Spectrophotometer:
  • Hydrogen gas lamp
  • Mercury lamp
• Visible Spectrophotometer:
  • Tungsten lamp

Monochromator

• Can handle all light or polychromatic light.

Sample Cells

• UV Spectrophotometer: Quartz (crystalline silica)
• Visible Spectrophotometer: Glass

Applications of Spectrophotometry

• UV Spectrophotometry: Used for compounds with chromophores (molecular groups containing electronic systems that absorb energy in the UV region).
  • Applications: Protein, Amino Acids (aromatic), Glucose Determination, Enzyme Activity (Hexokinase)
• Visible Spectrophotometry: Used for colored compounds. If the compound is colorless, a complexing agent can be added to form a colored complex.
  • Applications: Niacin, Pyridoxine, Vitamin B12, Metal Determination (Fe), Fat-quality Determination (TBA), Enzyme Activity (glucose oxidase)

Determining Sample Concentration

1. Measure the maximum wavelength.
2. Create a standard curve.
3. Measure the sample.
4. Convert the sample's absorbance using the standard curve.

Measurement Requirements

• Visible Spectrophotometry:
  • Sample in solution absorbs visible light (350-770 nm).
  • Sample solution must be clear and colored.
  • Solvent does not absorb visible light.
• UV Spectrophotometry:
  • Sample in solution absorbs UV light (180-350 nm).
  • Molecules of the compound have double bonds or nonbonding electrons (n-$π^<em>$, $π-π^</em>$, n-$δ^*$ transitions).
  • Clear solution, may be colorless.

UV-Vis Spectroscopy

• Definition: A spectroscopic analytical technique using near-ultraviolet electromagnetic radiation (190-380 nm) and visible light (380-780 nm) with a spectrophotometer.
• Involves significant electronic energy within the analyzed molecule, thus more useful for quantitative analysis than qualitative.
• Qualitative analysis is used for secondary or supporting data.

Qualitative Analysis in UV-Vis Spectroscopy

• Two main determinations:
  1. Checking the purity of the UV-Vis spectrum.
  2. Determining the maximum wavelength.
• Maximum wavelength determination is based on calculating the shift in maximum wavelength due to the addition of groups to the parent chromophore system.

Infrared (IR) Spectroscopy

• A method observing the interaction between molecules and electromagnetic radiation with wavelengths of 0.75–1,000 μm or wave numbers of 13,000–10 cm-1.
• Widely used in industrial analysis and research labs to provide useful information for qualitative and quantitative analysis and help determine the structure of a compound. In other words, it helps applying the structural formula of a compound.