How to avoid trouble

Abstract Some hardware tips



  • Buy HPLC grade solvents, including H2O.
  • Record lot no.
  • Sparge all solvents, particularly water
  • Check H2O for Organic contaminants.
    • Pump 100ml of H2O through RP-18 column; then use MeOH gradient, UV-254.
    • Compare peak areas
  • Filter all buffers. (use 0.5 mm filters).
  • Add 100 ppm of sodium azide to aqueous buffers .

Sample preparation

  • Sample clean-up
    • Filter and centrifuge
    • Solvent Extraction
    • SPE (Solid Phase Extraction)
  • Use a sample solvent equal to or weaker in solvent strength than the mobile phase. 
    • Avoids precipitation
    • Improves resolution
  • Use a dilute solution if possible (1% or less in 20 or 50 μl loop).


  • For increased pump life, keep system pressure  below 2000 psi. 
  • Biggest enemy of pump is strong buffer.
    • Always wash out buffer thoroughly before shutting down.
    • Use dilute buffers (0.01 M or weaker).
    • Wash, sonicate (if necessary replace)pump seals every 6 months. Otherwise store in 50:50 MeOH : Water, replace every year.
  • Always purge the pump first when beginning with a new solvent system;
  • Dedicate a pump for use with solvent systems that are either polar or nonpolar.
  • Salt deposits at connections indicate leaks.
  • Never run a pump when it is dry.  The pistons and seals must first be wetted by priming the pump.
  • Methanol is the best choice for priming because it “wets” surfaces easily.
  • Air,dirt and buffers are your pump’s biggest enemies.


  • Purchase from major reliable manufacturer.
  • Test column characteristics.
    • A. Inert mix for channels ( toluene)        
    • B. Efficiency, use toluene-plate height should be 21/2 ´ (or less) the particle diameter.  If not return column.
    • C. Inject basic compounds( pyridine) to test for acidic sites.
    • D. Pore diameter, should be 3 times the solute diameter. (70-120 Å).
  • Minimize pressure surges and mechanical shocks.
  • Use column temperatures < 60˚C.
  • For overnight and storage, purge out salts and buffers.  Store in acetronitrile/water.
  • Store column (GPC, GFC, IEC) in recommended solvent.
  • To prevent sample adsorption :
    • Use guard columns.
    • Sample clean-up (filter, SPE, SFE).
    • Frequent column purge with strong solvent. Use hot water if buffers were used.
    • Regenerate column (reverse flush new columns).
  • To prevent chemical attack on columns :
    • Choose separation conditions more carefully
      (pH, Temp., Solvents)
    • Measure pH of sample (neutralize if necessary).

Dying column

Symptoms of a dying column

  • Pressure increase.
  • Tailing/Splitting peaks.
  • Loss in plate number.
  • Decrease in retention.
  • Pump pressure increases /Severe Tailing/Double peaks
    • Replace inlet frit.– filter cloudy samples
    • use in-line filters.
    • Check for precipitation at column head. Wash with hot water or strong solvent, 20 column volumes.

Detectors maintenance

  • Electrical problems - call manufacturer.
  • Old (burned out) lamp- replace.
    • Measure sensitivity regularly with standards, replace lamp before it burns out.
  • Leaks, air bubbles - sharp spikes.
  • Dirty cell window - low sensitivity (wash cell with 5% HNO3 for 20 minutes, flush with lots of H2O).
  • Do not clean columns through detector.
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