LCMS

• Luminescence 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
• 3 types of luminescence:

  • Fluorescence

  • Phosphorescence

  • Chemiluminescence
• fluorescence is when a beam of light is incident on

  certain substances, they emit visible light

  or radiations
• fluorescence starts immediately after the absorption

  of light and stops as soon as the incident

  light is cut off
• Phosphorescence is delayed fluorescence when light radiation is incident on

  certain substances, they emit light

  continuously even after the incident light

  is cut off.
• Fluorometry measures the fluorescence or the energy emission that occurs when

  a certain compound absorb electromagnetic radiation, become

  excited and then return to an energy state that is usually higher than

  their original level
• Primary Monochromator - selects the wavelength that is best absorbed by the solution to be

  measured
• Secondary Monochromator filters out fluorescence from stray light radiation, position at a right angle from the cuvette to eliminate potential

  interference from the excitation light
• advantages of fluorometry: increased sensitivity and specificity
• Quenching - quick disappearance of fluorescence
• Chemiluminescence is the production of light from a chemical reaction
• fluorophore is the atom or molecule that fluoroses/Fluorescence properties
• chemiluminescence 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
• chemiluminescence excitation energy comes from a chemical or

  electrochemical reaction
• Nephelometry and Turbidimetry - light scattering is a physical phenomenon that results from the

  interaction of light with particles in solution.
• Nephelometry measures the amount of light

  scattered in a particulate

  suspension at 90o angle
• Turbidimetry measures the amount of light

  blocked in a particulate

  suspension
• Nephelometry - Mercury arc lamp, Scattered light is measured
• The higher the turbidity, the smaller the

  quantity of light transmitted
• Turbidimetry - Tungsten lamp, Light transmitted is measured
• Nephelometry - Measured at 90 degrees, PMT is the detector
• Turbidimetry - Measured in straight line, Photocell is the detector
• Factors that affect scattered light (1) Particle size
• Factors that affect scattered light (2) Concentration of particles
• Factors that affect scattered light (3) Molecular weight of particles
• Factors that affect scattered light (4) Wavelength dependence
• Three Types of Scattered Light : rayleigh , mie , rayleigh debye
• rayleigh - Wavelength of light > particle size Light symmetrically scattered

  around the particle
• MIE - Wavelength of light < particle size
• rayleigh debye - Wavelength of light = particle size
• 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
• in MIE Light scatters backward but appears

  forward due to destruction out of

  phase background scatter
• in rayleigh light symmetrically scattered

  around the particle
• in rayleigh debye More forward light scatter
• Eluent - Fluid or substance that enters the

  column and moves the analyte
• Eluate – separated components
• Elution - The process of washing out a compound through a column using a suitable

  solvent
• mobile phases of chromatography :

  • Gas chromatography

  • Liquid chromatography
• gas chromatography - separating compounds based primarily on their volatility, is useful for compounds that are naturally volatile or can be easily converted into a volatile form
• Gas-Liquid Chromatography: based on partition