Chromatography

  • Created by: SamDavies
  • Created on: 02-01-19 18:52
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  • Chromatography
    • Paper chromatography
      • Solid phase: cellulose (polar)   Mobile phase: Any (usually H2O/EtOH mix)
      • Separates ink based on affinity of the colours to paper (cellulose)
    • Thin layer chromatography
      • Solid phase: silica (polar) Mobile phase: organic solvent
      • Rf value = the distance travelled by the compound / the distance travelled by the eluent
        • Increasing the polarity of the solvent will increase the distance of the compound travelled because there is now a higher affinity for the mobile phase
      • Not good for higher quantities of material
    • Column chromatography
      • Identical principle to TLC but allows for higher quantities of material
      • Solid phase: silica column Mobile phase: organic solvent
        • Most non-polar component will elute off first
    • HPLC
      • Uses reverse phase chromatography (inert non-polar stationary phase and polar mobile phase)
        • Starts with an aqueous eluent, adding more organic components at intervals until the eluent is 100% organic (gradient)
      • Solvent is pumped through a column with C18 silica with the sample added into the stream
      • Attached to a detector at the end which analyses drops of the sample OR to a UV-Vis spectrometer
      • Non-UV absorbing components will not be observed
      • Retention time: the time taken to reach the other end of the HPLC machine from injection
        • Rt will always be the same for the same compound provided the solvent system remains the same
    • LCMS
      • Couples HPLC with mass spec
      • LCMS is only quantitative in relation to standards
  • Gradient solvent systems change the polarity of the solvent as the sample is run through the HPLC machine
  • Isocratic solvent systems (i.e. the same solvent) produce broad peaks with lower max concentrations

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