Chromophores and Electromagnetic Radiation

Pharmaceutical Analytical Chemistry III (PA 303)

• Level 2 PharmD (Spectroscopy)
• Lecture 1, Dr. Bassam Shaaban (10/10/2023)

Contents

• Principles of spectrophotometry
• Light and radiation
• Electromagnetic spectrum
• Light as energy
• Interaction of photons with matter
• Absorption spectrum, characteristics and shifts
• Types of electronic transitions
• Chromophores and auxochromes
• Factors affecting absorption spectrum (pH, solvent)
• Quantitative spectrophotometry (laws of light absorption)
• Instrumentation (basic component of spectrophotometer)
• Applications of spectrophotometry

Analytical Chemistry

• Volumetric analysis (indirect method)
• Qualitative analysis
• Quantitative analysis
• Volumetric analysis
• Instrumental analysis (Spectroscopy, Electrochemical, Chromatography)

Instrumental Methods of Analysis (Physicochemical)

• Measuring physical properties quantitatively related to constituent concentration.
• Requires specific instruments for instrumental analysis.
• Uses a stimulus (light, heat, current, or voltage) applied to sample.
• Signal is processed, and analyzed, to give a readout (meter, plotter, or computer).

Spectroscopy

• Why do solutions/matter appear colored?
• Light (electromagnetic radiation) composed of electric and magnetic components.
  • EMR is made up of discrete packets of energy called photons or quanta.
  • EMR also has wave-like properties described by wave motion.

Wave Properties of Electromagnetic Radiation

• Light exhibits both wave and particle properties.
• Light waves travel at the highest known speed of 3 x 10⁸ m/s.
• Wavelength classification according to the wavelength (λ).

Wavelength

• Linear distance between the crests of two successive waves.

Units of Length

• 1 angstrom (A°) = 10⁻¹⁰ m = 10⁻⁸ cm
• 1 nanometer (nm) = 10 A° = 10⁻⁷ cm
• 1 micrometer (µm) = 10⁴ A° = 10⁻⁴ cm

Wavelength and Frequency

• Frequency (ν) and wavelength (λ) are inversely proportional. (λ=c/ν)
  • where c is the speed of light

Particle Properties of Electromagnetic Radiation

• Electromagnetic radiation consists of photons
• Photon energy is directly proportional to the frequency (E=hν)
  • where h is Planck's constant (6.63 x 10⁻³⁴J.s)

Electromagnetic Spectrum

• Ranges from very short to very long wavelengths.
• Includes gamma rays, X-rays, ultraviolet, visible, infrared, microwaves, and radio waves.
  • Each region has a unique range of wavelengths and frequencies.

UV-Visible Spectrum

• Region of the electromagnetic spectrum the human eye can perceive. (400 - 780 nm).
• Spectroscopic technique useful to identify and measure concentrations of solutions.

Color

• White light is passed through a prism, broken into a spectrum of colors (red, orange, yellow, green, blue, indigo, violet).

Color Wheel

• Visual tool for illustrating color relationships.
  • Primary colors: red, yellow, blue
  • Complementary colors: colors opposite each other on the color wheel

Interactions of Photons with Matter

• Three basic processes by which a molecule (or atoms) absorbs radiation:
• Rotational transitions
• Vibrational transitions
• Electronic transitions

Electronic Spectrum

• Molecules at room temperature are generally in their lowest electronic energy state (ground state)
• Upon interaction with UV or visible photons, an outer electron (valence electron) in the molecule can move to a higher electronic energy level (excited state).
• Energy change associated follows the equation: ΔE=Es-Eg=hv = hc/入.

Lecture 2, Dr. Bassam Shaaban, PhD (12-Oct-24)

UV-Vis Luminescence Spectroscopy

• Human eye can perceive narrow electromagnetic spectrum. ~400-800nm

• UV-visible absorption spectra can identify compounds and quantify concentrations of solutions.

Electron Interactions

Molecules are typically in their ground state. Excited molecules can relax through several pathways;

• Non-radiative processes:
  • Heat release
  • Chemical Reaction
  • Internal conversion / External Conversion
• Radiative process: Emission of photons
  • Fluorescence (fast)
  • Phosphorescence (slow).

UV-Vis Transitions

• σ electrons usually require high-energy photons, Absorption occurs in the far UV region
• π electrons are involved in absorption but need less energy, commonly the medium to high UV range.
• n electrons is weaker than the others, Absorption occurs in UV region.

Types of Electrons

• Closed shell electrons: not involved in bonding

• Covalent single bond electrons: high excitation energy, thus require UV energy

• Electrons in π orbital (double or triple bonds): medium range of energy.

• Paired non-bonding outer electrons (n electrons): low excitation energy.

Types of Electronic Transitions

• σ → σ (saturated compounds, require high energy photons)

• π → π (conjugated compounds, medium energy photons)

• n→π n electrons relax faster, low excitation energy.

Absorption Spectra

• Characterization spectra; plot of absorbance versus wavelength.

  • Shows the wavelength of maximum absorption(λmax)

• Line spectra: atomic spectra, sharp, single wavelength of absorbance (e.g., sodium).

• Band spectra: molecular spectra, combinations of vibration and rotation sub levels with maxium absorption(λmax)

Factors Affecting Absorption Spectra

• pH: spectra of compounds with acidic or basic groups is dependent on pH.

• Solvents: less polar solvents interact less strongly with solute than polar solvents, influence positioning of max.

Types of Spectrophotometers

• Single-beam spectrophotometers
• Double-beam spectrophotometers; more accurate, sample measured twice by the same components simultaneously by use of beam spitter

Components of Spectrophotometers

• Light source (Xenon and Mercury lamps depending on UV or Visible spectra)

• Monochromator (Filters or grating to select certain wavelengths)

• Sample cell (Transparent to wavelength of light of interested region, glass or Quartz cuvettes)

• Detector (measures intensity of emitted light, phototubes and photomultiplier tubes)

• Recorder (analog or digital recording of signal on paper or computer)

Applications

• Qualitative analysis (identification of compounds): based on unique spectra.

• Quantitative analysis (measuring the concentration of compounds): Beer-Lambert law (amount of absorption of solution is directly proportional to concentration)

Deviations from Beer-Lambert's Law

• Deviation from Beer-Lambert's Law is due to interactions and association between analyte molecules at high concentrations.

• Instrumental errors: Unmatched cells, unclean handling or optics can distort absorbance at high concentration, and stray light from non sample sources.

Spectrophotometric Titration

• Measuring absorbance during titration to determine the end-point.

Advantages of Spectrophotometry

• Applicability: many organic/inorganic compounds absorb light
• High sensitivity: to accurately measure concentrations of the micro molar range
• Selectivity: due to selective reactions, measurements, and mathematical treatment
• Accuracy / Precision: has good accuracy, relative errors of 0.1 - 2%
• Convenient : rapid and useful method

Disadvantages of Spectrophotometry

• Interference from other substances (only if end point depends on the absorbance).

Additional Notes

• Factors that can affect the absorption of light (spectra) ; pH Solvent Concentration
• Additional study information on the spectral characteristic of each analyte(substance).

Lecture 3, 10/19/2024

Contents

• Principles of spectrophotometry
• Light and radiation
• Electromagnetic spectrum
• Light as energy
• Interaction of photons with matter
• Absorption spectrum, characteristics, and shifts
• Types of electronic transitions
• Chromophores and auxochromes
• Quantitative spectrophotometry (laws of light absorption)
• Factors affecting absorption spectrum (pH, solvent)
• Instrumentation (basic components of spectrophotometer)
• Applications of spectrophotometry

Factor Affecting on Absorption Spectra

- pH
- Solvents

Lecture 4, 10/27/2024

Contents

• Principles of spectrophotometry
• Light and radiation
• Electromagnetic spectrum
• Light as energy
• Interaction of photons with matter
• Absorption spectrum, characteristics, and shifts
• Types of electronic transitions
• Chromophores and auxochromes
• Quantitative spectrophotometry (laws of light absorption)
• Factors affecting absorption spectrum (pH, solvent)
• Instrumentation (basic component of spectrophotometer)
• Applications of spectrophotometry

Additional Notes

• Principles of atomic and molecular spectroscopy

Fluorimetry Lecture

• Introduction to the basics of fluorimetry

  • Principles of photoluminescence

• Variables that affect fluorescence intensity

• Advantages / Disadvantages of Fluorimetry

• Instrumentation (components of a fluorimeter, including filters, light sources, and detectors)

• Practical aspects of multi-component analysis

• Understanding how to select the ideal derivative order for given measurements

Multicomponent Analysis

• Multicomponent analysis methodology
  • Analysis of binary mixture (via simultaneous equations or derivative spectroscopy)

Atomic Spectroscopy

• Principles of Atomic Absorption Spectroscopy (AAS) and Flame Emission Spectroscopy (FES)
• Characteristics and methods of determination