Air Bubbles Exiting the HPLC PUMP, Reasons Why.

Reasons For Air Bubbles Exiting The HPLC Pump:

• 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.

 Any of the above causes may contribute to valves not functioning properly or air being drawn into the HPLC system. Troubleshooting should begin with the easiest and obvious areas first. Check the condition of the low pressure tubing used to make the connections to and from the mobile phase bottles and pump. If it is kinked, twisted or damaged, replace it with new tubing. Check the fittings used (nuts and ferrules) for tightness and to insure they have been installed properly and are not leaking. Repair all leaks. Keep the system clean (it is easier to monitor and troubleshoot problems when it is clean). Replace any damaged fittings with new ones. Check the solvent pickup filters monthly to insure they are clean and not obstructed. Make sure the flow rate you are using is within the acceptable range for your degasser. 

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.

HPLC Maintenance & Repair Parts To Have on Hand for HPLC Systems

HPLC (UHPLC) systems are complex instruments which require periodic inspection, cleaning and maintenance. These tasks are critical to maintain the performance, reliability and accuracy of the instrument. If you have not done so already, I strongly recommend that you create formal standard operating procedures (SOP's) which address: (1) The frequency of when routine and non-routine maintenance procedures should be performed; (2) The types of maintenance and/or repair procedures used (e.g. piston seal replacement, A/I rotary valve seal replacement); (3) The exact step-by-step procedure to follow in performing these tasks and (4) The Performance Verification or Qualification steps and procedures which are to be performed to verify that any repairs made have been done correctly. *An instrument log book should be employed to document these procedures over time.

Periodic "General Maintenance" of the HPLC is one type of service procedure which should be scheduled at a set frequency (Example: Every 6 months) and will serve to provide a time to clean, inspect and repair/replace any parts which are worn due to normal use. Such routine HPLC maintenance is often referred to as a basic "Preventative Maintenance" service (or "PM Service"). Spare parts common to your HPLC system(s) should be on hand to perform these scheduled maintenance procedures as part of a normal PM service.

Here is a list of common parts that should be on hand for a "typical" HPLC system used in a pharmaceutical laboratory. Please consult the appropriate manufacture's product literature to determine the correct parts needed for your own HPLC system. This list is presented as a general guideline only:

• Capillary tubing, fittings (nuts and ferrules): Assorted fittings, usually made of 316 Stainless Steel, but could be made of polymeric materials. Always have spare precut and polished chromatography tubing of appropriate I.D. and lengths for use with your HPLC available at all times. Insure that the nuts and ferrules used are appropriate for your brand of HPLC system and the columns used as different manufacturers have different specifications for their fittings and ferrules. Many types are not interchangeable.

• Detector Lamps: At least one spare bulb of a type designed for your specific detector should be on hand. Note that some detectors use multiple lamps so you may need to have more than one type available for each detector. Some lamp bulb types (e.g. tungsten) can be safely stored and last for several years while other types, such as Deuterium bulbs, loose substantial energy after as little as 6 months. If you have several detectors of the exact same design, then there is often no need to stock multiple replacement bulbs for each one. Instead, stock enough bulbs to service one detector as it is unlikely you would see failure of more than one detector on the same day (an exception to this guideline is if you perform PM services on all of the instruments at the same time, then you may want to have multiple bulbs available).

• Pump Pistons: One set of spare new pistons should be kept on hand for each pump module. As with lamp bulbs, if you have several identical pumps, then there is often no need to stock multiple sets of pistons for each one. Stock only as many as you expect to use in one year. Clean and inspect the pistons during each PM for any signs of scratches or surface abrasions. Under routine use, pistons should only require general cleaning and last a long time before replacement is required (> 1 year). Mobile phases which contain high concentrations of salt buffers often accelerate this wear requiring more frequent replacement. *Always install new piston seals when replacing pistons.

• Pump Piston Seals: At least one set of spare new piston seals should be on hand for each pump module. Seals wear out more frequently than pistons. You should go through two or more sets of piston seals before you need to replace the pistons. If the piston seals leak, inspect the pistons for wear (replace with new ones or clean and reuse) and install new piston seals. Mobile phases which contain high concentrations of salt buffers often accelerate this wear.

• Solvent Pickup Filters: These are the large particle filters which sit inside your solvent or mobile phase bottles. They are often made from stainless steel or sintered glass with porous inlets (~10 to 30 micron) and can clog or become fouled over time (esp. when used with aqueous buffers). In some cases these can be cleaned using sonication (not sintered glass filters, only steel or polymeric!). Note: Sometimes it is most cost effective to replace them with new filters then clean and re-use them.

• Inline Frits/Filters: You may have an inline filter placed after your PUMP head, but before the column inlet to collect any remaining particulate matter. These filters can extend the lifetime of the entire HPLC system (esp. the A/S, A/I and Column), but will only do so if changed on a regular basis. Some manufacturers incorporate this type of filter into the design of their pump modules. An example of this can be found on the HP/Agilent brand model 1050, 1100 and/or 1200-series pumps. These have an inexpensive 10 micron PTFE frit installed in the outlet valve of the pump. This filter catches all of the normally occurring piston seal debris and larger mobile phase particles and should be changed every month. Other pre-filters are installed in cartridges just before the column inlet. These often overlooked pre-filters filters must be replaced about once each month to do their job properly. Keep plenty of spare filters on hand.

• Auto-injector Rotary Valve Seals: If you have an auto-injector, then a high pressure valve is probably used to switch the sample into the flow path for analysis. This valve will have one or more parts which require regular inspection, cleaning and periodic replacement. Mobile phases which contain high concentrations of salt buffers often accelerate this wear. The valve rotor seal is the most common part which requires replacement.

• Auto-Sampler Needle: A needle should last a very long time, but depending on the frequency of use and type of vial septa encountered it can require replacement at regular intervals. A good general guideline would be to keep one spare needle on hand for every 2-4 systems.

• Auto-Sampler Needle Seat: The needle seat often requires more frequent replacement than the needle due to repeated mechanical wear. A good general guideline would be to keep one spare needle seat on hand for each system.

• UV/VIS Detector Flow Cell: While not actually a required PM spare part, this one is worthwhile to have. If you employ a UV/VIS flow cell, then I always suggest you keep one dedicated spare flow cell on hand which matches the size and volume of the type you use in your instrument. A spare flow cell can prove to be very valuable as a troubleshooting tool if you believe that you have contaminated or clogged your current flow cell. A quick swap can answer the question and get you back to work quickly saving hours or days of lost time. *Note: This extra flow cell should be kept separate from all instruments for use as a tested spare only and not used for regular analysis.

If you have suggestions for other types of common HPLC spares to add to the list or to have on hand, then please let me know.