part 3

• Parameters in chromatography

  • Retention
  • Efficiency
  • Selectivity
  • Resolution
• Factors that can affect retention

  • Solubility / affinity of the component in the SP
  • Temperature of the oven
  • Column length
  • Flow rate of the MP
• Efficiency


  A measure of the separating power of a column
• Higher efficiency column


  Higher separating power, better resolution
• Theoretical plate


  Equivalent to the points at which separation occurs (ie when analyte interact with the SP)
• Selectivity (α)


  Measures the difference in the retention behaviour of two components in a column
• Higher α

  Greater difference between the two components in their retention behaviour on the column, better separation between the peaks
• Resolution (R)


  Most important parameter, measures the degree of separation between two peaks
• Baseline resolution to achieve in chromatography: R = 1.5
• Temperature programming


  Gradually increasing the oven temperature (at a programmed rate) to improve the resolution of the peaks
• Compounds with low retention


  Lower temperature for well-resolved peaks
• Compounds with high retention


  Higher temperature for faster elution and sharp peaks
• Internal standards method


  Commonly used quantitative method in GC to correct for system variability (eg fluctuations in injection volumes)
• Internal standards method


  1. Add a compound (which is different from the analyte of interest) of known concentration into the sample

  2. This compound is called internal standard

  3. The ratio of the internal standard compound and that of the analyte is fixed regardless of the actual injection volume
• What makes a good internal standard?

  • Must have similar properties and structures as the analyte so that it can be eluted in the same region as the analyte
  • Can be well-resolved from the other components in the test sample
  • Must not be present in the test sample