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.

Do your HPLC Methods Meet Good Chromatography Fundamentals? HPLC Training: RETAIN, SEPARATE and RESOLVE

When an HPLC or LC-MS method is not developed properly, it may not be selective for the sample and may not show any retention on the column. When this happens, everything injected may elute out at the same time and appear to be 100% pure . *These types of errors are easy to spot by anyone with formal training and experience in chromatography concepts (note: "years" on the job are not the same thing as years of practical knowledge and/or formal training in the technique. We routinely provide consulting services to clients with 10 or more years on the job performing chromatography analysis, but whom have not received any formal training during this time and make errors of this type).

Developing HPLC Methods which follow good chromatography guidelines and fundamentals should be key goals of HPLC method development. When developing an HPLC ("UHPLC") method, you must develop an analysis method which is selective for the compound of interest. 'Selectivity' is the most important variable to focus on when developing methods. Your method must demonstrate that it can: (1) Retain; (2) Separate and (3) baseline Resolve all peaks present (and any possible impurities or related substances), in a reliable and repeatable way. Failure to demonstrate that your HPLC method meets these basic requirements AND is selective for the sample being analyzed means your method is invalid.  

*You may be surprised to know that many HPLC methods (including some published papers and "Validated" Methods) do not meet these basic requirements. In this case, knowledge is truly power. If you have the practical knowledge and understanding of this technique, you will be able to easily spot these invalid methods. Make sure you review other methods as part of your training. Never assume because someone else published it or "did it that way", that it is valid. It may not be. An average of 20% of the methods I review do not meet these basic requirements and are invalid.

• Do your HPLC methods meet these requirements? 
• Can you demonstrate to others, who are knowledgeable in the technique, that your method follows good fundamentals? 

You should be able to demonstrate knowledge of these basic principles and have confidence in them.

Proper HPLC method development training must include and stress the following three practical, fundamental concepts of Retain, Separate and Resolve:

• Demonstrate that using your HPLC Method, that the sample is RETAINED on the Column. *Screen many columns to find the best one, early in the process. For most modes of chromatography, you do this by first estimating then measuring the column void volume. How do you know if it is retained long enough? Next, you calculate the K prime (Capacity Factor) of your sample to insure it meets basic chromatography guidelines (or regulations). * K prime > 1.5 (or > 2.0 for most regulated environments). Note: While retention is required, K prime is not applicable to SEC modes of chromatography.

• When satisfactory retention is achieved (K prime), begin optimizing the method to SEPARATE and baseline RESOLVE all compounds. This is achieved using proper HPLC Method Development techniques plus the practical knowledge and experience which comes from good training. Please refer to these two linked articles for more information; "Modern HPLC Method Development Tips (PART I)" and "Modern HPLC Method Development Tips (PART II)".

HPLC PEAK Fronting and Tailing, Common Reasons For It

All users of HPLC need to know and be familiar with the correct terms used to describe non-Gaussian shaped peaks. Two of the most common undesirable peak shapes, peaks that show "Fronting" and peaks that show "Tailing" indicate problems with the HPLC method.  A quick refresher on why you may observe an HPLC peak front or tail on the chromatogram follows. 

Peak FRONTING: First, let us define what peak fronting looks like. The leading edge (front) of the peak is vertical, straight up and non-Gaussian in shape. This sharp increase in signal is easy to spot. 

Common Reasons for Peak FRONTING:

• Poor sample/peak capacity. In other words, too low a K prime (not enough retention on the HPLC column) resulting in no chromatography taking place. To solve this problem you must develop a proper HPLC method which first retains the compound(s) of interest, holds them long enough to obtain an acceptable K prime and resolve them away from other peaks, then elutes them off the column.

• Injection Solution Too Strong:Your sample(s) should be dissolved in the mobile phase and not in a solution that is "stronger" in elution strength than the mobile phase. Example: If you method is 100% aqueous, do not inject the sample in a solution with organic solvent. Follow fundamental good chromatography guidelines.

• Column Fouling / Overloading of sample. When the HPLC column is overloaded with sample, the peak shape will show fronting. Decrease the injection volume and/or concentration, as appropriate, in 10x graduations until the peak shape is normal.

• Saturation of the Detector: Just as with overloading the column the peak shape may change, overloading the detector's measuring range may also result in saturation of the signal and loss of accuracy. Decrease the injection volume and/or concentration, as appropriate, in 10x graduations until the peak shape is normal and back on-scale.

Peak TAILING: First, let us define what peak tailing looks like. The trailing edge (tail) of the peak slowly drops off towards the baseline and  is non-Gaussian in shape. For those with GC experience it appears similar to a peak that "bleeds" and continues to interact with the column for an extended period of time.

Common Reasons for Peak TAILING:

• Flow path Diffusion (from extra-delay volume). Poorly swaged fittings/connectors, a column with a void, incorrectly sized capillary connection lines may all contribute to peak tailing. Optimize the flow path, column and connections.

• pH dependence for ionizable compounds. If the sample is easily ionized and the difference between the pka of the sample and the mobile phase is less than 2 pH unit, tailing may result. Being sure to work within a safe pH range for your column, increase or decrease the mobile phase pH to be > 2 pH units away from the sample's pka to reduce tailing.

• Type 'A' silica or heavy metal contamination of the support. Many older style column supports did not use ultra-pure, heavy metal free packing material. These material often interacted with the sample on the column resulting in changes in retention, The use of more modern type 'B' or 'C' packings has eliminated many of these problems.

• Residual silanol groups present on support. As with the earlier type 'A' supports, non fully end-capped supports with residual silanol groups often resulted in secondary, extended retention effects. Use of more modern, fully end-capped, ultra-high purity packing materials (and/or mobile phases which better address these residual groups) often allow Gaussian peak shapes without the need for many additives.

• Column Fouling / Overloading of sample. When a column is not washed of all retained material after each analysis, it may build up over time and change the surface chemistry of the support. This may lead to changes in retention, especially delays in both binding and elution. Wash, regenerate or replace the column to solve.

You may also be interested in reading a related article; "Two Common HPLC Problems and their Causes (Sudden changes to either the HPLC Backpressure or Peak Shape)".

A Case of Changing Solution pH. Formic Acid Stability in Solution (Methanol)

Real life examples help to better illustrate problems that I am called in to troubleshoot for clients. As a professional scientific consultant, many of my clients have spent months (sometimes years) trying to solve an analytical problem on their own before I am brought in to make the diagnosis and propose a solution. Many years of working in a wide range of scientific fields allows me to identify problems quickly and efficiently saving clients the most money and allowing them to resume work on their projects.

This was the case during a recent consult for a major cannabis testing laboratory. They were having a great deal of difficulty obtaining reproducible results for their analytical testing screens (14 compounds in their analysis with a need for repeatable and accurate results). Variations from 25% to 50% were observed run-to-run over the course of seven days. They assured me they were doing everything in the same way. To begin the troubleshooting process, we started by looking at the actual data gathered and the actual method(s) used to acquire the data. These were evaluated to see if they followed good practices and techniques, also to make sure they had SOP's in place which were clear. Good SOP's must include enough detail to allow anyone reviewing them to prepare samples, standards and/or solutions in the exact same way. Additionally, the HPLC instrumentation was checked and tested to verify it was performing as designed.

After reviewing their training and methodologies on-site, a number of areas of concern were quickly identified. One of the most likely reasons for the variation in values over time was found to be caused by a common mistake in the preparation of mobile phase solutions for the HPLC system. To save time, the client's scientists prepared all organic solvent solutions in advance (~ one month or more), then filtered and stored them at room temperature. For example, their solutions of 0.1% formic acid in HPLC grade Methanol were pre-mixed and stored in glass one liter bottles. These bottles were then put aside, for an average of one month before use. This finding proved key as someone with proper HPLC training would be aware of a well known problem when formic acid is left in pure organic solvent, especially methanol, over time (less so with ACN). Briefly, the formic acid content degrades quickly over time and is often found to be only half of what it was initially after just three or four days (If you have not done so already, this is a simple and useful experiment to run in your lab, monitoring the acid level by titration, not with a pH meter, over time at room temperature in methanol)! This degradation continues over time reducing the amount of acid in solution. If the acid is added to the solution to enhance ionization (i.e. LC-MS; LC-MS/MS) or provide acidification to maintain the sample in a fully ionized form, then as the level of acidification decreases, so does the solution's ability to maintain it. In other words, your HPLC method may change over time (resulting in an in-valid method).

•  I have always promoted the importance of making and using freshly prepared mobile phase solutions (daily), especially where any aqueous solutions are used (to prevent degradation of additives and/or bacterial or fungi growth). However, this precaution does not normally apply to many pure organic solvents, but there are a few very important exceptions to this, formic acid and methanol in this example. 

Changes were made to their SOP's to insure that future solutions of formic acid in methanol were not prepared in advance, but instead, fresh on the day needed only. This coupled with a few basic improvements to their column washing, equilibration and overall training resulted in %RSD of only 0.3% for future analysis runs.

 
As a side note, I have been asked why solutions of formic acid in methanol are sold commercially for HPLC use? I have no answer to this, but respectfully remind everyone that just because something is offered for sale, does not mean it should be purchased. Ask yourself if the item is appropriate for your application? It may not be suitable for your use or application. 

BTW: Please be sure to flush your HPLC system of all organic acids (e.g. acetic, formic) after use and do not leave them in the HPLC system overnight. Even 1% levels of organic acids may be corrosive to stainless steel.