QUAILE SEMIS

• Spectrophotometry


  Measurement of light intensity in a narrower wavelength

  
• Photometric Measurement


  Measurement of light intensity without consideration of wavelength

  
• Spectrophotometric Measurement


  Measurement of light intensity in a narrower wavelength

  
• Beer's Law


  States that the concentration of a substance is directly proportional to the amount of optical density (absorbance) or inversely proportional to the logarithm of the transmitted light
• Light Source


  • Provides the energy that the sample will modify and attenuate by absorption
  • Provides incident light for the system
  • Continuum & Line Sources
  • Incandescent Tungsten or Tungsten-Iodide Lamp (most common, visible and near IR region)
  • Alternatives: Mercury arc, Deuterium lamp, Hydrogen lamp (for UV Spectrum), Mercury arc, Nernst glower and Globar (for IR Spectrum)

  
• Entrance Slit


  • Minimizes stray light
  • Prevents the entrance of scattered light
• Monochromator


  • Used to isolate an individual wavelength of light
  • Degree of isolation of the wavelength is affected by the monochromator and the width of the entrance and exit slits
  • Colored glass filters, prisms and diffraction gratings

  
• Exit Slit


  • Controls the width of the light beam (band pass)
  • Allows only a fraction of the spectrum to reach the sample cuvette
  • The narrower the bandpass, the greater the resolution

  
• Sample Cell


  • Also known as cuvette or analytical cell
  • Holds the solution of which the absorption is to be measured
• Photodetector


  • Converts transmitted radiant energy into an equivalent amount of electrical energy
  • Kinds: Photocell, Phototube, Phototransistors / Photodiode, Photomultiplier Tube

  
• Photomultiplier (PMT)


  • Most common type (visible and UV)
  • Amplifies radiant energy (200x sensitive)
  • Excellent sensitivity and rapid response
  • Can detect very low level of light and quick burst of light

  
• Read Out Device


  • Displays the output of the detection system
  • Galvanometer / Ammeter / LED Display

  
• Atomic Absorption Spectrophotometry


  • The amount of light absorbed is proportional to the concentration
  • Ground State → Excited State
  • The excited atom then returns to the ground state, emitting light of the same energy as it absorbed
• Light Source (Atomic Absorption Spectrophotometry)

  • Electrodeless Discharge Lamp (Consists of bulb filled with argon and the element to be tested, Radiofrequency generator excites the element)
  • Hollow-Cathode Lamp (Consists of an evacuated gas-tight chamber containing an anode, a cylindrical cathode, and an inert gas, such as helium or argon, Separate lamp is required for each metal)

  
• Beam Chopper


  • Modulates the hollow cathode light beam
  • Produces pulses of light
• Nebulizer


  • Delivers a fine spray of sample containing metallic ion into the flame / cylinder
• Atomizer


  • Sample cell of the instrument
  • Dissociates the solution into its neutral and individual atoms
  • Flame atomizer (A fuel gas (acetylene) with an oxidizing agent (compressed air) is burned to product the flame)
  • Electrothermal Atomizer (Graphite Furnace, Flameless Atomic Absorption)
• Burner Head


  • Made of Titanium
  • Must not be made of compositions detected by Atomic Absorption
  • Burner Heads Used: 10 cm single slot burner (Air / Acetylene Flames), 5 cm single wide slot burner (Air / Acetylene Flames), 5 cm single slot burner (Nitrous Oxide / Acetylene Flames)
• Monochromator & Photodetector (Atomic Absorption Spectrophotometry)


  • Monochromator (The light absorbed by the sample is in pulses, thus the light it emits is also in pulses, Isolate desired emission line from other lamp emission lines, Entrance and exit slits)
  • Photodetector (Photomultiplier Tube)

  
• Luminescence


  It is based on an energy exchange process that occurs when certain compounds absorb electromagnetic radiation, become excited, and return to an energy level lower than or equal to their original level
• Fluorescence


  Photoluminescence; The emission is basically immediate and therefore generally only visible, it the light source is continuously on

  
• Phosphorescence


  Photoluminescence; It is characterized when materials can store the light energy for some time and release light later

  
• Chemiluminescence


  The emission of light is created from a chemical or electrochemical reaction and not from absorption of electromagnetic energy
• Fluorometry


  Molecular Luminescence Spectroscopy; Measures the amount of light emitted by a molecule after excitation by electromagnetic radiation; It is 1000 times more sensitive than spectrophotometry; It is more specific
• Basic Set-Up of Fluorometry

  • Light Source, Attenuator, Secondary / Emission Monochromator, Photodetector, Read-out Device

  
• Light Source (Fluorometry)


  • It is the source of the short wave and high energy light; Mercury and Xenon Arc
• Attenuator


  • Intensifies the light from the light source
• Secondary / Emission Monochromator


  • It is placed on a right angle from the cuvette to avoid incidence light from reaching the detector

  
• Photodetector (Fluorometry)


  • Converts light energy to its equivalent electrical energy; detects the fluorescence light; Photomultiplier Tube is the most commonly used photodetector

  
• Quenching Phenomenon


  This phenomenon when the excited state of the molecule loses some of its energy by interaction to other components of the reaction system
• Chemiluminescence

  The emission of light is created from a chemical or electrochemical reaction and not from absorption of electromagnetic energy; No excitation radiation is needed; Applied in most serological techniques; Subpicomolar measurement; Speed, ease of use and simple instrumentation; Background signals

  
• Electrophoresis

  Separation of charged compounds based on their electrical charge; The process of separating the charged constituents of a sample by means of an electrical current
• Amphoteric

  • A substance that can have a negative, zero, or positive charge depending on conditions
• Anion


  • Negatively charge; Migrates to the anode; Anode - It is the positively charge electrode
• Cation


  • Positively charge; Migrates to the cathode; Cathode - It is the negatively charge electrode
• Factors affecting mobility of particles in electrophoresis


  • Net charge of the particle
  • Size and shape of the particle
  • Strength of the electric field
  • Chemical and physical properties of the medium
  • Electrophoretic temperature
• Power Supply


  • Supplies constant current or voltage in the system; Driving force; Voltage depends on the ionic strength of the buffer
• Buffer


  • Used to provide ions that carry a current and to maintain the pH at a relatively constant value; pH (acidic = binds H+ = ampholyte becomes positively charge, cation = migrates to the cathode; basic = loses H+ = ampholyte becomes negatively charge, anion = migrates to the anode); Ionic strength (LOW I.S = more charge will be carried = faster mobility; HIGH I.S = less charge will be carried = slower mobility)
• Support Media


  • A network of interacting fibers or a polymer that is solid but traps large amount of solvent in pores or channel inside; Must not interact with the analyte; Cellulose Acetate, Agarose Gel, Polyacrylamide Gel
• Detecting System


  • Electrophoretogram (Result of electrophoresis consisting of separated strands of a macromolecules)
  • Direct Observation
  • Staining (Specific for one chemical group)
  • Radioactive Dye (Iodine - 125)
  • UV Visualization
  • Densitometer (A thermometer is a device that measures the degree of darkness (the optical density) of a photographic or semitransparent material or of a reflecting surface)