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 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).
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.
- 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.