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Chromatography Column selectivity - a - PowerPoint PPT Presentation


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Chromatography Column selectivity - a. Elution volume: V m =mobile phase interstitial volume V s =stationary phase volume V R is the retention volume of an unretained compound + an additional volume of mobile phase required to elute a compound

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slide1

Chromatography

Column selectivity - a

  • Elution volume:
    • Vm =mobile phase interstitial volume
    • Vs=stationary phase volume
    • VR is the retention volume of an unretained compound + an additional volume of mobile phase required to elute a compound
    • Large values of K means a compound has a high affinity for a stationary phase
    • K is strongly temperature dependant - Changes a
    • Changing K changes column selectivity
      • Change the nature of the mobile phase - pH, polarity, ionic strength nature of the gas
      • Change the nature of the stationary phase - pore size of gels, surfaces of adsorbants, the liquid phase, ion exchangers
      • Change the nature of the solute - charge on an amino acid, complexing agents, derivatize analyte
slide2

Chromatography

    • Elution volume …
      • VR (or tR) can be varied under conditions of constant K by changing the ratio of Vm to VS
    • Capacity factor
      • Small k’ values mean little retention
        • If tR-tm=0, solute elutes with the mobile phase front because the stationary phase is not used or under utilized
      • Large k’ values mean analysis times are long which can result in broad peaks that may be difficult to detect
      • Good values for k’ ~ 1.5 - 4
    • Column efficiency, column selectivity and capacity factor can be combined with resolution
slide4

Chromatography

column column capacity

efficiency selectivity factor

slide5

Chromatography

Time required for a given analysis

slide6

Chromatography

Plot of tR and R versus k’

slide7

Chromatography

  • Additional relationships
      • K is temperature dependant: change k’ by changing temperature
        • In GC, temperature programming can be used to control k’, thus retention times and affect resolution
      • K is mobile phase dependent: change k’ by changing mobile phase which is particularly effective for HPLC
      • As a approaches 1, Nreq becomes large
        • To improve R, adjust N or k’
slide8

Chromatography

  • Qualitative analysis often makes use of elution times measured from the time of injection
    • One may also use retention volumes: VR = tR x f, where f is the volume flow rate
    • Retention times are characteristic of a given compound on a specific column used under a given set of conditions including mobile flow velocity, pressure, mobile phase and temperature
    • A very useful detector for qualitative analysis is a mass spectrometer
  • Quantitative analysis generally makes use of peak areas
    • Peak heights can vary if conditions are such that retention times vary
      • Peaks change peak width with retention time
      • Constant areas with varying peak width produce varying peak height
  • Peak shape depends on the shape of the isotherm associated with a peak
    • The isotherm represents how K changes with the concentration of analyte in the mobile phase at a particular temperature
      • So far we have assumed K is independent of concentration, that is there is a linear isotherm
      • Non-linear isotherms produce distorted peak shape
      • One way to look at this is to examine how the nature of the stationary phase changes as the concentration of analyte changes with conc.
slide9

Chromatography

The effect of isotherm shape on peak shape and retention time as a function of sample size