- Pump Cavitation: When the pump pressure fluctuates wildly up and down, at very low pressures, this is often due to 'pump cavitation'. It is caused by a loss of priming inside the pump (Air, instead of liquid is in the pump's flow path). The HPLC pump should be primed with fresh, degassed mobile phase (following proper procedures) to restore smooth, stable flow. Often, this can be accomplished using the pump, set to a high flow rate, to draw liquid from the bottles. In cases where the pump is not strog enough, manually priming the low pressure lines using a syringe (~ 20 mL) filled with mobile phase and opening (or disconnecting) a fitting at the pump's outlet may aid in priming the system. Note: Depending on the configuration of your HPLC system, to fully prime an HPLC pump, you may need to run 20 or more mLs of solution through EACH channel. Please keep this in mind every time you use the system and every time you prepare or change a mobile phase solution. This article on baseline/pressure fluctuations may assist you in troubleshooting.
- Loose Connections: If one or more of the low-pressure fittings (nuts and ferrules) which secure the Teflon tubing to the pump (or vacuum degasser) are damaged or loose, air may enter the system resulting in bubbles. Most pumps use plastic finger-tight style fittings 1/4-28 (or 5/16-24). The threads are soft and can be deformed. When access to these fittings is difficult, sometimes the fittings are left loose and will allow small amounts of air to be drawn in. A build up of salts and/or buffers on the exposed fittings can also allow air into the system (and the presence of deposits on the fittings indicates poor maintenance and a LEAK !). Inspect the tubing and fittings used for proper type, seating depth, wear/condition, cleanliness and/or damage. Replace parts as needed and re-install using the correct amount of torque.
- Flow Rate Too High, Too Low or Not Enough Degasser Equilibration Time: Degassing efficiency is directly related to the flow rate. Lower flow rates increase the residence time of the mobile phase in the degassing membrane or tubing, improving the gas removal. Higher flow rates provide less time for gas extraction and result in lower degassing efficiency (which equals bubbles in the outlet line). Check with the manufacturer regarding the optimal flow rate range for your degasser to insure you are working within an acceptable range. Allow enough time for the degasser to reach its set-point and stabilize before use. If the degasser is not operating properly or is unable to "keep up" with the flow rate, then bubbles may be frequently observed in the outlet lines.
- Choice of Mobile Phase Liquid: The miscibility of the liquid is also important. If the new mobile phase is not compatible with the previously used mobile phase, pump cavitation may result. Always flush the pump with an intermediate liquid that will dissolve in both the old and new fluids to flush them out before introducing the new mobile phase solution. (such as pure water or IPA, as applicable). The solubility of air (gas) in the specific solution used also affects the efficiency of the vacuum degasser. Aqueous solutions usually hold less gas than popular organic solvents (though air bubbles can be harder to "push" through in water). The amount of dissolved gas inside the liquid relates directly to the time needed to reduce it to acceptable levels for use in HPLC. Be sure to allow enough time to properly degass the new solution.
- Dirty or Obstructed Solvent Pickup Filters (Bottle filters): Bottle filters should be cleaned or replaced at regular intervals, following routine maintenance SOPs. When they become fouled or obstructed, a vacuum may form as the liquid is drawn into the system. This may result in air being sucked into the tubing or through a fitting (remember that the low pressure Teflon tubing used to connect the bottles to the degasser and pump is porous and allows gas to diffuse through it). The pickup filters should not obstruct the normal flow of solvent (typically they are 10-20 u in porosity). * a quick troubleshooting tip to rule out an obstructed solvent pickup filter is to temporarily remove the filter from the bottle. Observe the back-pressure on the pump to see if it increases and priming is restored. If so, the filter may be clogged. Always replace the filter with a fresh, clean filter and never operate the HPLC without the solvent filters installed.
- A Sticking Check Valve: The pump's inlet and outlet check valves must function perfectly, all of the time, to maintain proper flow and pump function. If an inlet check valve is not fully closing properly to seal off the high pressures generated inside the pump, then the pump will not be able to maintain pressure or flow. Inspect the check valve. Remove and clean it, per the manufacturer's guidelines (often this involves placing the check valve assembly in a beaker with solvent such as IPA and sonicating for 20 minutes to remove any residues. If cleaning fails to restore proper valve function, then replace the check valve with a new one.
- Worn Pump Piston Seals (or Pistons): When the piston seals begin to leak, air is allowed into the system. Pump piston seals require regular replacement (they are normal wear items). Scratched or worn pistons may also result in leaks with air getting into the system. Inspect and Test them both for pressure tightness on a scheduled basis or anytime you suspect a problem. Flush the pump with a suitable liquid, then run a high-pressure test to determine if they pass or fail the manufacturer's leak tightness and high pressure tests. Be sure to perform a physical inspection too.
- Contaminated or Obstructed Pump Outlet Filter: Most HPLC pumps have a small disposable outlet filter installed at or near the pump outlet line (Note: In the case of most Agilent brand HPLC pumps, a small PTFE filter may be found at the outlet valve or inside of the prime-purge valve). These filters should be replaced at regular intervals (monthly is strongly recommended), especially if any aqueous buffers or solutions are used (a they contribute to contamination). Contaminated pump outlet filters may result in a number of pressure instability problems. Abnormally high back-pressure during operation OR when vented to waste are indications it is obstructed. Regular scheduled replacement is the best way to prevent lost time and reduce system contamination.
Has your degasser module been professionally cleaned and serviced within the last 5 years? Are any degasser errors being generated? Is the vacuum degasser making any unusual sounds? Is liquid being emitted from the vacuum pump exhaust port? If any of the answers to these questions are 'yes', then have the HPLC vacuum degasser professionally diagnosed for problems so that repairs can be made to restore function.
