Chromatography & spectroscopy

• • Chromatography is a technique used to separate components with similar physical properties from a mixture
• • To do this a mobile phase is passed over a stationary phase & each compound has a different affinity for each phase
  • A phase is a physically distinct form of a substance
• • The stationary phase slows compound movement & the more interactions there are the more it is slowed down
  • Allowed compound to move at different rates
• Separation:

  • Solid stationary phase --> separate by adsorption as molecules bind on to the surface
  • Stronger adsorption = slower
  • Liquid stationary phase --> separate by relative solubility as molecules dissolve into the phase
  • Greater solubility = slower
• Thin-layer chromatography:

  • A stationary phase of silica gel (SiO2) or alumina (AlO3) is coated onto a plate of glass or plastic
  • A sample is added to a starting point on the plate which is then placed into a liquid solvent which will get drawn up
  • At the end the height of the solvent is marked - the solvent front
  • Perform thin-layer chromatography on some amino acids
• Rf values:

  • An Rf value shows how far a component has moved relative to the solvent front
  • Rf = distance moved by component / distance moved by solvent front
  • This can identify a component as it will always have the same Rf values, however:
  • Similar compounds may have similar Rf values
  • A suitable solvent is needed to separate the different compounds
• Gas chromatography:

  • A stationary phase of a solid silicone polymers or a liquid long chain alkane is coated inside a capillary tube
  • A sample is injected & vapourised before being pushed through the capillary column by a carrier gas ( mobile gas)
  • An inert / unreactive gas such as He or N2
• Retention time:

  • The detector at the end records how long it took for a component to pass through the column ( retention time)
  • the area below a peak is proportional to the concentration
  • The retention time can help identify the component but:
  • chemicals can have the same retention time, peak shape & detector response
  • One peak can be hidden by others with the same retention time but a greater concentration
  • To improve results chromatography is often paired with mass spectroscopy
• Nuclear magnetic resonance (NMR):

  • Nuclear magnetic resonance can be used to analyse a molecular structure in detail
  • Usually with the isotopes 1H, 13C, 19F & 31P
• NMR analysis:

  • A strong magnetic field (electromagnet)
  • Low-energy radio frequency radiation
  • MRI scanners (magnetic resonance imaging) work in the same way to produce images of soft tissue in hospitals
• Resonance:

  • Nuclei can be promoted to a higher energy spin state by providing energy to match the gap -known as excitation
  • Supplied by the low energy radio frequency radiation
  • The larger the energy change, the larger the frequency required
  • When the nuclei later drop to their original lower energy state they emit the same amount of energy - known as relaxation
• Resonance --> the cycle of excitation & relaxation that occurs & continues as long as the frequency equals the energy gap
• Nuclear shielding:

  • Nuclear shielding occurs when weak magnetic fields from the electrons surrounding a nucleus counteract the applied field
  • This is also caused by nearby electrons as well
  • The extent shielding depends on the density of electrons in other atoms & groups giving atoms different environments each with different resonance frequencies
• Chemical shift:

  • The position at which a nucleus absorbs energy along the NMR spectrum is known as the chemical shift
  • The shift is relative to a reference signal
  • Each TMS ( tetramethylsilane) molecule has 12 equivalent protons which give it a distinct NMR signal which is given the value of 0ppm
  • TMS is used as it is unreactive & its volatility allows it to be removed from the sample at the end
• NMR solvents:

  • NMR is carried out in solution & to avoid unwanted signals deuterated solvents are used
  • A common solvent used is CDCL3 with its carbon-13 peak removed from the spectra
  • D represents the hydrogen isotope Deuterium
  • Deuterium has 2 nucleons & so has no spin & so signal on an NMR spectrum
• Carbon -13 NMR:

  • C13 makes up around 1.1% of all carbon atoms & as it has an odd number of nucleons it has a residual magnetic spin
  • The chemical shift indicate the chemical environment of the carbon atoms present
  • If there is an electronegative atom/group there is a significant chemical shift
• Carbon -13 spectra:

  • Spectra produces shows:
  • The number of environments = number of peaks
  • The type of environment = chemical shift of a peak
• • There are 4 main types of carbon atom that absorb over different chemical shift ranges
  • The chemical shifts may also be outside of these ranges, depending on the solvent, concentration & substituents
  • The chemical environment of a carbon atom is determined by the position of the atom within the molecule
• • Carbon atoms that are bonded to different atoms or groups of atoms have different environments & will absorb at different chemical shifts
  • If 2 carbon atoms are positioned symmetrically within a molecule, then they are equivalent & have the same chemical environment
  • They will then absorb radiation at the same chemical shift & contribute to the same peak