pharm analysis - EMR/UV spectroscopy

• how is electromagnetic radiation classified?

  • can be classified in order of decreasing wavelength = electromagnetic spectrum.
  • Higher wavelength = Lower frequency = Lower energy
• wavelength increases to the right

  
• why can gamma rays be dangerous to tissues?

  Ionisation. They can eject electrons.
• what are light waves?

  Light waves are electromagnetic : made of moving electric (E) and magnetic (B) fields vibrating 90 degrees to each other at speed of light. They are moving all the time. 

  It is the energy from these fields (electromagnetic) that makes the wave move. The wave generates a photon.
• Lights are made of photons and they have different energy depending on its wavelengths.
• what is the frequency of a wave?

  The frequency of a wave is the number of complete cycles of the wave that pass a point in a given time.
• what is the wavelength of a wave?

  length of one cycle

  
• what is the equation involving frequency and wavelength?

  Speed of light formula

  
• how does an increase in wavelength affect the frequency?

  Decreases frequency.

  • For light c is the same/constant (3 x 108 m/s , vacuum).
  • Thus λ increases as frequency decreases (and vice versa) to keep c the same.
• • Energy is also related to frequency:
  • h is the Plank’s constant
  • energy is directly proportional to the frequency
  • Hence: high frequency = low wavelength = high energy

  
• what are photons?

  Particles of light.

  Light is energy carrying waves which also behave like particles travelling discrete units called photons (each has a unique packet or quantum of energy)
• what can happen when a photon hits something like a solution or any matter?

  Absorption, reflection, or transmission.

  • the energy can be transferred - the photon energy can be absorbed by the material so there's interaction with the material
  • when photons hit something, EMR waves interact like other waves
• energy from each photon: E=hc/λ
• what does spectroscopy involve?

  Interactions between matter and electromagnetic radiation. Involves absorption, emission, or scattering (less common - when light hits the material light can get scattered) of EMR by substances.
• dynamic light scattering...

  If light hits particles like nanoparticles in a protein formulation it can get scattered → dynamic light scattering can be used to determine the size of those particles.
• if a photon hits a sample, for example, you can get:

  Absorption

  • Selective removal of certain frequencies by transfer of energy to atoms or molecules
  • Electrons promoted from lower-energy (ground) states to higher energy (excited) states.
  • To do that the energy of exciting photon must exactly match the energy difference between the ground state and one of the excited states of the absorbing species.

  Emission

  • Once the electrons are promoted you could get emission
  • Electromagnetic radiation is produced when excited particles return to lower-energy levels or the ground state.
• total internal energy in a molecule is the sum of energy from what? (spectroscopy)

  1. electrons moving around
  2. vibrations between the molecule’s own atoms
  3. rotations of the molecule in the bonds
• Common spectroscopic techniques which rely on use of  EMR in the pharmaceutical analysis include:

  • UV-Vis absorption - electrons excited
  • Infra-Red (IR) absorption - vibrations excited
  • Fluorescence emission - electrons returning back
• what happens when photons hit a molecule?

  • a molecule/atom changes its energy state by absorbing or hitting energy that is equal to the energy difference between ground E0 and excited E1 state.
  • the energy emitted back and returns to ground state

  A) absorption

  B) emission

  
• electronic states...

  • Each electronic state is subdivided into vibrational states. And then further subdivisions into rotational states.
  • each energy level is discrete but has further vibrational and rotational levels which are also discrete.
  • excitation of a molecule can thus be electronic, vibrational and rotational
• Electronic states -> If electrons are only moving in the vibrational state and they only excite the vibrations = IR spectroscopy
• electronic states in an image

  A) more

  B) short

  C) less

  D) visible

  E) vibrational

  F) longer

  
• what provides the energy to excite electrons?

  Photons from light excite electrons from the ground state to the excited state
• sigma bonding molecular orbital...

  formed by electrons in 2 S orbitals – forming single bonds. Electrons are most close to the nucleus. And so the pull is very high and this means they require more energy.
• π (bonding in double or triple bonds) molecular orbital...

  formed by electrons in p orbitals forming double or triple bonds. Not so close to the nucleus so requires less energy.
• n (non-bonding) atomic orbital...

  found on e.g. nitrogen, oxygen as lone pairs (these are just lone pairs of electrons)
• energy level diagram - with anti-bonding orbitals

  • σ and non-bonding electrons can move to σ*
  • π can move to π*
  • Only π to π* and n  to π* are in the right region (low enough energy) for any use in pharmaceutical analysis.  The others are in far UV where air interferes.

  A) antibonding

  B) highest

  
• what are anti-bonding orbitals?

  Orbitals with higher energy than bonding orbitals.

  When a bonding orbital (HOMO) is created there is also created a corresponding anti-bonding orbital (LUMO) that is normally unoccupied but lies at a higher (less stable) state.
• When you create a sigma bond (which is a low state), you also have to create another anti-bonding orbital because energy is conserved. And so each sigma bond is associated with a σ* which is a higher bond.
• HIGH ENERGY = SHORTER WAVELENGTHS = HIGH FREQUENCY
• what values of absorption are not used as much in analysis?

  • <200nm absorption
  • σ to σ* n to σ*  would require photons of λ below 200nm to get the right level of excitation energy. In this far UV region molecules in medium (air/solvent) will have similar transitions so practically the spectrum can not be measured easily.
• what values of absorption are most common for drug analysis?

  Only π to π* or  n  to π* are in this region.  The molecule must contain pi bonds (unsaturated) or atoms (oxygen, nitrogen, halogen) with non-bonding orbitals. In reality UV absorption is mostly restricted to drug molecules with conjugated bonds (π to π* ).
• what is the UV-visible spectrum of a drug?

  plot of absorbance against wavelength - when the electrons get excited you get absorption
• • Most drugs having π bonds absorb UV light.
  • Double bonds absorb energy and so these drugs will need to be protected from the light. For degradation purposes for stability of drugs this is important as it makes the drug vulnerable to the photooxidation and photo-degradation.
• The closer you move to shorter wavelengths (when doing an assay) the more interference you start getting from the solvent.
• Drugs such as menthol has no double bonds thus very little absorption in UV >200nm.
• what type of drug molecules have weak absorption in UV >200um?

  drugs such as menthol as it doesn't have any double bonds and so has very little absorption
• What type of bond absorbs UV light?

  Conjugated bonds and π bonds
• what allows a molecule to have strong absorption of UV light?

  lots of double bonds + highly conjugated system
• recording the absorbance means you can work out what?

  Concentration - How much drug is present.

  Higher absorbance = the more drug is in solution that you’re analysing.