Air Bubbles Exiting the HPLC Vacuum Degasser. Reasons Why

A common question we are asked to solve relates to why air bubbles might be observed exiting out of an HPLC vacuum degasser module  (where the mobile phase leaves the degasser ports to go to the pump heads and/or gradient valve)? Troubleshooting and answering this question is most easily accomplished if you first have a solid understanding of the HPLC flow path, how to make proper connections and are familiar with performing routine maintenance on the HPLC system. 

• Key Point: HPLC systems utilize Teflon low-pressure tubing to transfer the mobile phase (solvents) from the mobile phase bottles to the HPLC pump. The Teflon lines are permeable to gas in the atmosphere. Gas is diffusing through the plastic tubing used to transport your solvents. This is one of the reasons why we purge the entire flow path of the HPLC system before use, each day. Overnight, gas has diffused into the system so we start by flushing (purge) the mobile phase from each bottle, through the degasser, through each channel all the way to the pump head, to waste.

To find the reason why air bubbles may be observed exiting the HPLC vacuum degasser module, we examine the flow path.
 
Common Reasons For Air Bubbles Exiting The HPLC Vacuum Degasser Include:

• Loose Connections: If the low pressure fittings (nuts and ferrules)  which secure the Teflon tubing to the degasser are damaged or loose, air may enter the system resulting in bubbles. Most vacuum degassers 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 (such as found on many of the generic small benchtop degasser which use the micro-chambers or the HP/Agilent model G1379-series). Inspect the tubing and fittings used for proper seating depth, wear and/or damage. Replace parts as needed and re-install using the correct amount of torque.

• Flow Rate Too High 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. 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.

• Choice of Mobile Phase Liquid: 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.

• 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).

• Vacuum Degasser Damage: HPLC Vacuum degasser modules, like most other component parts of your HPLC system break down over time and require professional diagnostic testing, cleaning and repair. Under ideal conditions, most inline electronic vacuum degassers require diagnostic testing and cleaning or repair every 4 to 5 years. *Many show signs of contamination or failure before that time. The internal vacuum tubing becomes contaminated and worn over time. The vacuum pump is an electromechanical part which is exposed to all of the mobile phase additives and solvent vapors during use. Other internal component parts such as vacuum valves or restrictors may also become contaminated or worn over time. The vacuum degassing membranes (or tubing) themselves can stretch from use and wear out over time. The vacuum chambers may be exposed to incompatible chemicals or over-pressured resulting in internal leakage. Certain chemicals may also attack and even dissolve the degassing membranes causing more internal damage and contamination of the mobile phase. These devices do not have any "contamination" detection alarms and the vacuum sensors sometimes become damaged over time leading to false vacuum levels being reported. Never rely on the module's built-in error alarm system as proof of compliance (no more than you would the reported flow rate shown on the computer screen. It must be measured to be known). Regular professional HPLC degasser testing and service are required to maintain the modules and meet compliance requirements.

 Any of the above causes may contribute to air being drawn into the degasser 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 degasser. 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. Replace any damaged fittings with new ones. Check the solvent pickups 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 degasser making any unusual sounds? 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. 

Additional Information:

• "Agilent / HP Brand Vacuum Degasser Troubleshooting Tips, Errors and Repairs"; https://www.hplctools.com/agilent_degasser_error_troubleshooting.htm

• "Waters Alliance and Acquity Brand UPLC Vacuum Degasser Troubleshooting Ti[s and Errors"; https://www.hplctools.com/waters_degasser_error_troubleshooting.htm

Gradient Mixing Test For Your HPLC Pump (Step Gradient)

The most popular type of gradient pumping module used to perform HPLC analysis utilizes a low pressure mixing valve in their design. These valves are electronically controlled and proportion the amount of mobile phase from one of several solvent channels into a mixer for introduction to the pump head (*the solenoid valves used for this are sometimes called gradient proportioning valves). They provide random access to multiple solvents (e.g. 4) for method development and column flushing. The mobile phase solutions are mixed at low pressure before entering the high pressure side of the pump head (where they undergo compression). This design requires only one high pressure pumping head and can allow for very high mixing accuracy (often 0.1% per channel) of the mobile phase. This allows for the formation of mobile phase gradients over time which greatly aid in resolving samples apart on the column.

The gradient proportioning valves need to be tested along with the other parts of your HPLC system on a regular basis to insure they are operating within the manufacturer's specifications. They should also be tested anytime you suspect a problem may be present. One quick way to check the operation of two of the valves is to use a tracer compound and STEP gradient to monitor their operation. You can set up a method to perform this test as suggested below.

QUICK GRADIENT COMPOSITION TEST:

Bottle A = 100% DH20;
Bottle B = 0.1 % Acetone in DH20 (*Acetone is the tracer compound);

Flow Rate = 1.000 ml/min;
Column = No column. Install a restriction capillary in place of the column to obtain a backpressure of > 60 Bars;

Detection = 265nm (10 nm bandwidth) UV;

STEP Gradient Program:
    0 to 2.00 min, 0 % B
    2.01 min, 20% B
    4.01 min, 40% B
    6.01 min, 60% B
    8.01 min, 80% B
  10.01 min 100 % B
  12.01 min 20% B
  14.00 min 20% B

Note: If the delay volume (dwell volume) of your system is large, then you may want to adjust the time values shown to LARGER values (i.e. 2 minutes delays are used in this example, but 5 or even 10 minute delays between steps may be more appropriate if your system has > 1 ml dwell volume.

Running the above method should result in a signal trace which shows a step-wise rise to 12.00 minutes (as the acetone concentration increases). The edges of the "steps" should be sharp and the risers should also be close to vertical. The final step change which starts at 10.01 minutes shows a linear gradient change back down to the 20% B level. This line should not have any bumps or dips in it and should transition smoothly back down. The height of the baseline at this point should match the height seen between 2.01 and 4.00 minutes (same 20% B). The height of the proportional steps (e.g. 20, 40, 60, 80) should also be the same. You can use your CDS to measure these height values.

Another useful aspect to view is the S/N ratio at each step. Use your CDS to establish noise windows within each range (e.g. 2.50 to 3.5 minutes). This data is useful when comparing the performance of the pump at different intervals.

If you observe deviations in the height of the proportional steps or dips in the lines, these can be caused by leaking or sticking check valves as well as leaking or sticking gradient proportioning valves. *If you have a quaternary pump, be sure and test all four of the valves used (2x per test).

Lastly, the above example is a generalized method and may or may not be applicable to your specific HPLC pump. Be sure and customize a test method which takes into account the pressure ranges, flow rates, delay volume, mixing volume, and number of low pressure channels used in your pump.