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

LC-MS Contamination? Another Possible Cause. Are your Mobile Phase Bottles and Pick up Filters Clean ?

One of the more common LC/MS problems I am asked to help solve deals with contaminated LC-MS or LC/MS/MS systems. Over time, many systems will become contaminated with a wide variety of plasticizers, detergents, salts, metals and ion pairing agents that routine source cleaning will not remove. Often, these compounds are introduced to the system through the tools used (e.g. pipettes) chemicals, solvents, mobile phase additives or even the samples themselves. "Dirty" samples sometimes persist inside the system long after the analysis work is complete, leaving material in poorly maintained injection valves but also through the use of poorly washed / contaminated and fouled HPLC columns. Even the modern inline HPLC vacuum degasser has proven to be a source of contamination. 

In addition to the above mentioned sources of contamination, another more obvious source of contamination should always be addressed early in the process of cleaning the system. Specifically, the glass mobile phase bottles and the associated solvent pickup tubing and solvent pickup filters used with them. Contamination in these areas may directly infuse the system with undesirable material. Good cleaning and maintenance practices must be maintained to reduce this source of potential contamination. 

As a general guideline, we shall not place our mobile phase reservoir bottles in any type of dishwasher or wash them using any dish soaps. These may leave a residue easily detected by even the weakest mass spectrometer. Avoid contamination by purchasing high quality glass bottles with vented caps to keep dust out. If rinsing with organic solvents (and/or freshly prepared and filtered high resistance water) does not clean them, you can try a Nitric Acid rinse (up to 30%) followed by a neutralizing wash in 2M Sodium hydroxide. Follow-up with many rinses of HPLC Grade water (or LC/MS grade), oven drying, then re-fill with an appropriate mobile phase. Don't forget to replace those solvent pickup filters too. While many 316 SS pickup filters can be cleaned, most of the sintered glass style filters are designed to be disposed of (not cleaned or put in an ultrasonic cleaner!). So periodically dispose of the glass types and install new filters and fresh mobile phase into those recently cleaned bottles (before you start looking for the source of contamination in the more expensive parts of the instrument, clean or replace the filters). - Please don't re-contaminate an expensive HPLC or LC/MS system and invalidate your methods and data because you skipped replacing a $10 part. Keep commonly used spare parts in-stock and always maintain a clean system.

Vacuum Pressure Units Conversion Table

Several of the questions I receive each week by email deal with scientific calculations or conversion of various units. One popular request relates to the conversion of micrograms, ppm and percent. Several years ago to address this question, I posted a table of weight to ppm units ("Conversion Factors microgram, nanogram, ppm, ppb and percent") which has proven to be very popular.

Because of the large number of vacuum pumps attached to HPLC and MS systems, another common conversion question relates to vacuum units. Due to the different applications and regions of the world, the desired unit often varies. It is for this reason that I develop unit conversion tables as I find these tables provide for a convenient way to print out and/or keep handy in a binder for future reference. Widespread computer use coupled to freely available page reader software (e.g. Adobe PDF) provides another means to store useful information as a pdf file too. I present this "Vacuum Pressure Units Conversion Table" in a viewable and an optionally available downloadable form [click HERE to download].

VACUUM PRESSURE UNITS CONVERSION TABLE:

*Some of the more commonly used values are shown in boldface type. ** Absolute Vacuum..

% Vacuum	Torr (mm Mercury)	kPa abs	Inches of Mercury	Micron	PSI
0.0	760.0	101.4	0.00	760,000	14.7
1.3	750.0	99.9	0.42	750,000	14.5
1.9	735.6	97.7	1.02	735,600	14.2
7.9	700.0	93.5	2.32	700,000	13.5
21.0	600.0	79.9	6.32	600,000	11.6
34.0	500.0	66.7	10.22	500,000	9.7
47.0	400.0	53.2	14.22	400,000	7.7
50.0	380.0	50.8	14.92	380,000	7.3
61.0	300.0	40	18.12	300,000	5.8
74.0	200.0	26.6	22.07	200,000	3.9
87.0	100.0	13.3	25.98	100,000	1.93
88.0	90.0	12	26.38	90,000	1.74
89.5	80.0	10.7	26.77	80,000	1.55
90.8	70.0	9.3	27.16	70,000	1.35
92.1	60.0	8	27.56	60,000	1.16
93.0	51.7	6.9	27.89	51,700	1.00
93.5	50.0	6.7	27.95	50,000	0.97
94.8	40.0	5.3	28.35	40,000	0.77
96.1	30.0	4	28.74	30,000	0.58
96.6	25.4	3.4	28.92	25,400	0.49
97.4	20.0	2.7	29.14	20,000	0.39
98.7	10.0	1.3	29.53	10,000	0.193
99.0	7.6	1.0	29.62	7,600	0.147
99.87	1.0	0.13	29.88	1,000	0.01934
99.90	0.75	0.1	29.89	750	0.0145
99.99	0.10	0.013	29.916	100	0.00193
99.999	0.01	0.0013	29.9196	10	0.000193
100	0.00	0	29.92	0	0

An Often Ignored HPLC & LC/MS Contamination Source. Did you check your Vacuum Degasser?

The introduction of electronic vacuum degassing / degasser modules to the liquid chromatography industry a few decades ago has introduced several new problems which were unknown years ago when we sparged our mobile phase solutions with high-purity helium gas. One of these problems relates to how the electronic vacuum degassing modules themselves can contribute to contaminating your HPLC or LC-MS system.

Before using an HPLC vacuum degasser module, please review all of the information and advice supplied by the manufacturer of your specific degassing module. The composition of the internal degassing tubing has changed a great deal over the past decades resulting in increased degassing efficiency, but also changes in mobile phase chemical compatibility. Some popular solvents may be incompatible with some models in your lab. Make sure you know exactly which types of vacuum chambers and plastics are used in your specific instrument(s). Degassing modules must be operated, cleaned and maintained the same as your other important instruments. When they are not operating properly and/or are contaminated, they should be serviced as soon as possible or risk further contamination and damage to your system ($$$).

•  Link to a pdf document with pictures showing common contamination and damage observed while inspecting HPLC Vacuum degasser modules for our clients. Have you had your HPLC vacuum degasser(s) professionally evaluated for damage? [ https://www.chiralizer.com/examples_of_degasser_contamination.pdf ].
  

In a previous post ["Inline HPLC Degassing Modules"] we discussed the convenience that these devices have brought to our laboratories, but also the extra training requirements (such as cleaning and flushing the vacuum channels every day and routine servicing every 2 to 5 years) which must be undertaken to use them successfully. When the operational guidelines for the use of these products are ignored, these devices can contribute to the contamination of your HPLC and /or LC-MS system. The internal wettable surfaces of each degassing chamber contain plastics (examples of plastic used: Teflon, Teflon AF, Tefzel and/or Peek are the most common types of used). To effectively remove gas from the mobile phase, the liquid must pass through plastic tubing (or across membranes) placed in a vacuum, for a period of time which is long enough to allow a portion of the dissolved gas in the mobile phase to diffuse through the degassing tubing/membrane and out the exhaust port of the degassing chamber. The degassing tubing (most use tubing) should have the maximum chemical compatibility possible while allowing it to also be porous enough for the gas alone to diffuse through the walls of the tubing under vacuum. These requirements usually result in some type of fluoropolymer tubing (Variants of Teflon or Teflon AF) being used as they have broad chemical compatibility plus can be formed with controlled pore sizes for the effective removal of gas, not liquid, through the tubing walls. However, there are exceptions to this and the plastic(s) used may NOT be chemically compatible with all liquids used in chromatography applications. Depending on the plastic degassing tubing used, the tubing may swell, fail or even dissolve into the mobile phase solution! Be sure and check the chemical compatibility chart offered by the degassing module manufacturer for compatibility with your mobile phase and ALL additives used before using the instrument. Some examples of incompatible chemicals on the lists of many instrument vendors include: THF, Chloroform, DCM, strong acids or bases, Hexanes and Sodium Azide. Use of incompatible solvents or additives may result in complete failure of the degasser module plus contamination of the entire instrument flow path. We have seen many degasser systems which were used with (or stored in, w/o proper flushing) strong acids show corrosion of the metal parts inside the chambers (SS fittings and connectors) forming piles of rust and salts which were carried through the vacuum system resulting in damage to the system and flow path contamination. *Please do not risk it. Be aware of which chemicals may pose a risk with your system. For example: The use of many fluorinated solvents may dissolve most types of tubing when Teflon AF is used for degassing.

• Note: We have seen an increase in the use of various perfluorinated solvents, esp with LC/MS systems. This has resulted in severe degasser damage plus MS source contamination (e.g. HFIP and Ethoxynonafluorobutane). Most perfluorinated solvents are not compatible with vacuum chambers which contain Teflon AF. They may dissolve the degassing tubing, resulting in the destruction of the degasser chambers and contamination of the vacuum system and mobile phase (IOW: the complete HPLC system flow path). Additionally, we commonly see ion-pairing reagents such as TFA and TBA "sticking" to the plastic used in these modules causing a leaching of material over long periods of time (again, most obvious on an MS system where you can "detect" it in the background signal). These ion-pairing agents must be thoroughly flushed out of the flow path to reduce contaminating the entire system over time. *A strong wash solution with a little acid (formic) alternated with a wash containing some base (ammonium) often helps in this regard. Wash cycles of over 12 hours are often needed to remove these compounds and see improvement (It may take much longer...). In some cases we must replace some or all of the internal parts of the degassing module to eliminate the contamination. Always remove any HPLC column from the flow path (to avoid re-contaminating again) and replace with a new one, once the contamination has been removed. It is for this reason that we should avoid the use of strong ion-pairing reagents in any LC/MS system, as they often contribute to very high background signals and long term contamination. *Helium sparging should be considered for such applications.

Reversed phase HPLC applications which use highly aqueous mobile phases may under some circumstances result in high rates of pervaporation of the water vapor into the degasser module resulting in condensation of the water into the vacuum system (Unlike the older Teflon material used, the newer Teflon AF formula is more permeable to water vapor). Once liquid enters the vacuum pump, severe damage has already occurred and failure of the vacuum system soon follows. *If you ever notice liquid exiting through the vac pump's exhaust port, turn off the HPLC system and have the degasser module professionally serviced. 

Another common problem seen when aqueous solutions are used in an in-line vacuum degasser are that of algae and bacterial growth. Most often observed in systems left unused for a period of time or which are not periodically flushed out with organic solvents. Growth inside the low pressure tubing and even inside the vacuum chambers has been known to contaminate the entire flow path of an HPLC system. Replacement of the tubing and internal chambers usually resolves the problem.

Buffers / Additives: Just as with the rest of your HPLC system, any buffer salts, acid, bases or additives which are left in the system (even overnight) can damage it. This is true of the vacuum degasser module too. Please be sure to flush all of the vacuum degassing chambers of any salts or buffers when not in use.

For normal phase applications, high concentrations of n-Hexane may cause contamination or damage to a degasser attached to an HPLC or LC/MS system. Some types of degassers are not compatible with Hexane. The ultra high evaporation rate of hexane coupled to the advanced materials found in the degassing tubing or membranes may result in the hexane condensing on the outside of the internal degassing tubing of the degasser and then aspirated into the vacuum system (causing damage). The contaminants are then transported back through the tubing walls into the solvent stream (your mobile phase).

If your HPLC's vacuum degasser fails to achieve vacuum, has liquid exiting the vacuum pump exhaust port (or exhaust tubing) or shows an error (e.g. Leak Error, High RPM, makes loud noise, a Yellow or Red light on an HP/Agilent system or "Degasser Hardware Fault" / "Degasser High Leak Rate" messages often seen on Waters brand systems), then your entire HPLC system may be out-of-compliance - because the degasser is broken. Have the vacuum degasser professionally cleaned and repaired so you can put the system back online. Do not assume that only the vacuum pump has failed, as replacement of the pump alone often results in failure of the replacement pump soon after (due to contamination and other problems incorrectly diagnosed). The true cause of the failure must be correctly diagnosed and repaired first, and this is something best left to professionals.

Our professional HPLC degasser repair shop receives many types of degasser modules with leaking or ruptured vacuum chambers. These directly contribute to mobile phase contamination as any seal failure in these normally "dry" systems results in liquid contaminating the vacuum system which in-turn sends contaminated liquid and vapors back into the HPLC mobile phase stream. *Note: If you are using your HPLC degasser with Mass Spec detector, then the resulting mobile phase contamination may contaminate not just your column, but MS source too (costing a great deal of money to decontaminate). Please, at the first sign of trouble, have the degasser professionally diagnosed, cleaned and repaired. For more information on having your degasser professionally diagnosed and repaired with fast turnaround at a fraction of the price charged by most instrument vendors, please refer to this link: "HPLC Degasser Repair Service" [ http://www.chiralizer.com/hplc-degasser-repair.html ]