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Study Notes: Achieving Good Separations

What can we say to make you a better chromatographer? You should remember that HPLC is an art as well as a science. We have covered some of the science in this section but the art must come from practice, experience and something called common sense.

The keys to good separations are as follows.

  • Know the chemistry of the components in the samples, and in the mobile and stationary phases.
  • Obtain a good application from a manufacturer and work with that and modify it to suit.
  • Pay great attention to detail -
    • no leaks in the system
    • mobile phase(s) fresh and degassed
    • no particulate matter in the system (use a pre-column filter and replace it regularly)
    • no column ‘poisons’ in the system (use a guard column to adsorb out the nasties)
    • use very high quality reagents and Milli Q or similar water
    • follow the manufacturer’s and application instructions
    • do not cut corners and do not be in a rush.
  • Always wash the column with the starting mobile phase before commencing the run and with the final mobile phase at the end of the run.
  • Look after that column.
  • Cover the mobile phase container(s) to exclude dust.
  • Pay attention to temperature.
  • Keep your eye on flow rates and pressures during the run.
  • NEVER run out of mobile phase.
  • Choose the correct wavelength for the detector.
  • Check and change the detector globe regularly.
  • Adjust the ‘gain’ on the chart recorder so that the peaks are not too small nor run off the top of the trace.
  • Adjust the baseline so that it sits correctly on the chart.
  • Use a reliable chart recorder.
  • Be exacting with injection volumes - use the same technique to inject every time.
  • Wash out the injection syringe every time.
  • Be careful in the making of the mobile phases to ensure consistency.
  • Be kind to your pumps and have them serviced regularly.
  • Have a clean and tidy work area.
  • Clean dust from the outside of the HPLC and all components.


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