2ND SEM

• Fluorescence


  • Delayed fluorescence
  • When light radiation is incident on certain substances, they emit light continuously even after the incident light is cut off
• When a beam of light is incident on certain substances, they emit visible light or radiations
• Luminescence


  • It is the emission of light by a substance
  • Occurs when an electron returns to the electronic ground state from an excited state and loses its excess energy as a photon
  • Three types: Fluorescence, Phosphorescence, Chemiluminescence
• Fluorescence vs Phosphorescence

  Fluorometry measures the fluorescence or the energy emission that occurs when a certain compound absorbs electromagnetic radiation, becomes excited, and then returns to an energy state that is usually higher than their original level
• What is Luminescence?
• Luminescence starts immediately after the absorption of light and stops as soon as the incident light is cut off
• Chemiluminescence


  • It is the production of light from a chemical reaction
  • Reactions are oxidation reactions of luminol, acridinium esters, and dioxetanes characterized by a rapid increase in intensity of emitted light followed by a gradual decay
  • The excitation of the substance does not involve electromagnetic radiation and no monochromators are needed, instead the excitation energy comes from a chemical or electrochemical reaction
  • Light signal is measured against a completely dark background
• How to measure fluorescence?

  1. Light source: Mercury vapor lamp or xenon arc lamp

  2. Excitation/Primary Monochromator selects the wavelength that is best absorbed by the solution to be measured

  3. Cuvette

  4. Emission/Secondary Monochromator filters out fluorescence from stray light radiation and is positioned at a right angle from the cuvette to eliminate potential interference from the excitation light

  5. Photodetector
• Fluorometry


  • Advantages: Increased sensitivity (1000x more sensitive than spectrophotometric methods), Emitted radiation is measured directly, Increased specificity by selecting the optimal wavelength for both absorption and fluorescence
  • Disadvantages: Very sensitive to environmental changes, Quenching- quick disappearance of fluorescence, Changes in pH affect electron availability, Temperature changes the probability of loss of energy, Contaminating chemicals or a change of solvent may change the structure, UV light used for excitation can cause photochemical changes
• Chemiluminescence


  • Advantages: Subpicomolar detection limits
  • Speed
  • Ease of use
  • Simple instrumentation
  • Disadvantages: Impurities can cause a background signal that degrades sensitivity and specificity
• Nephelometry and Turbidimetry

  1. Light scattering is a physical phenomenon that results from the interaction of light with particles in solution

  2. Unlike fluorescence emission, the wavelength of the scattered light is the same as that of the incident light
• Chemiluminescence


  1. The excitation of the substance does not involve electromagnetic radiation and no monochromators are needed, instead the excitation energy comes from a chemical or electrochemical reaction

  2. Light signal is measured against a completely dark background
• Three Types of Scattered Light

  • Wavelength of light > particle size: Rayleigh
  • Wavelength of light < particle size: Mie
  • Wavelength of light = particle size: Rayleigh Debye
• Nephelometry vs Turbidimetry

  • Nephelometry measures the amount of light scattered in a particulate suspension at a 90º angle
  • Useful method to determine the concentration of solutions that contain particles too large for absorption spectrometry
  • Turbidimetry measures the amount of light blocked in a particulate suspension
  • Decrease in light transmission
  • Amount of light blocked depends not only on concentration but also on size
  • Sampling handling becomes critical
• Factors that affect scattered light: Rayleigh, Mie, Rayleigh Debye
• Chromatography


  Is an analytical technique commonly used for separating a mixture of chemical substances into its individual components, so that the individual components can be thoroughly analyzed
• Basic Components of Chromatography

  • Mobile phase or carrier: Gas or liquid
  • Solvent moving through the column
  • Stationary phase or adsorbent: Solid or liquid
  • Column – holds the stationary phase
  • Eluate – separated components
  • Eluent: Fluid or substance that enters the column and moves the analyte
  • Elution: The process of washing out a compound through a column using a suitable solvent
  • Analyte: Mixture whose individual components have to be separated and analyzed
  • Retention time or factor: The time it takes for a compound or analyte to elute
  • Chromatographic techniques may be classified according to their mobile phase: Gas chromatography, Liquid chromatography
• Chromatographic techniques classified by their mobile phase

  • Gas chromatography
  • Liquid chromatography
• Chromatography


  Separating compounds based primarily on their volatility
• Gas Chromatography: separating compounds based primarily on their volatility
• Gas Chromatography is useful for compounds that are naturally volatile or can be easily converted into a volatile form
• Columns in Gas Chromatography: Packed columns or Capillary columns
• Types of Gas Chromatography

  • Gas-Liquid Chromatography
  • Gas-Solid Chromatography
• Components of Packed columns

  • Glass or stainless steel (packed)
  • Thin-fused silica (capillary)
• Components of Packed columns

  Filled with inert particles such as diatomaceous earth or porous polymer or glass beads coated with a nonvolatile liquid (stationary) phase
• Retention time or factor: The time it takes for a compound or analyte to elute
• Basic Components of Gas Chromatography: TWO DETECTORS - Thermal conductivity and Flame ionization detector
• Gas Chromatography makes it easier to recover a sample compared to Liquid Chromatography
• Liquid stationary phase in Packed columns

  Must be nonvolatile at the temperatures used, thermally stable, and must not react chemically with the solutes to be separated
• Gas Chromatography uses lower temperatures for separation achieving better separation of thermolabile compounds
• Liquid Chromatography Types of separation technique: Adsorption, Partition, Steric exclusion, Affinity, Ion-Exchange
• Liquid-Solid Chromatography: Competition between the sample and the mobile phase for the adsorptive sites on the solid stationary phase
• Liquid-Liquid Chromatography: Separation of substances according to their solubility in an organic/non-polar solvent and in an aqueous/polar solvent
• Steric Exclusion: Use of a resin for covalent attachment of anions or cations onto it
• Ion Exchange: Widely used for the separation of proteins, peptides, and nucleic acids
• Affinity Chromatography: Utilizes the specific interaction between one kind of solute molecule and a second molecule immobilized on a stationary phase
• Types of molecules

  • Proteins
  • Peptides
  • Nucleic acids
• Sample injectors

  1. Used to introduce the sample into the path of the mobile phase that carries it into the column

  2. Loop injector is the best and most widely used with high reproducibility and used at high pressures
• Affinity chromatography

  • Most selective type of chromatography employed
  • Utilizes the specific interaction between one kind of solute molecule and a second molecule that is immobilized on a stationary phase
  • Examples: antigen and antibody, enzyme and substrate, receptor and ligand, protein and nucleic acid
• Thin-Layer Chromatography

  Uses pressure for fast separations, controlled temperature, in-line detectors, and gradient elution techniques