Troubleshooting a GRADUAL HPLC PUMP PRESSURE INCREASE OVER TIME (When PURGING, DURING an ANALYSIS or when FLOW is DIRECTED TO WASTE)

A fully equilibrated column at a constant temperature and flow rate should result in a stable back-pressure over time (~1% variation). Have you observed slowly increasing HPLC system back-pressure readings, even when you are fully equilibrated and everything should be stable? Read on to find out why this may be happening...

First, you must know what are "normal" or expected values for:

• HPLC Pressure (and normal changes over time);
• Baseline changes (e.g. drift, equilibration or normal rise/fall);
• Peaks (e.g. Sample peaks vs. Valve position peaks, spikes or Noise);
• Retention time delays (due to a leak, gradient delay, fouled column etc).
  

  vs. those that result from an actual hardware faults. To operate any HPLC system, you must learn how to identify these. It will take many years of hands-on practical experience.

If you know what to look for, the HPLC system will provide you with clues when something is wrong. "Pressure" is one of those clues... Learn to always pay special attention to the system back-pressure and watch for signs of trouble. Pressure should change in a predictable way.

• HPLC system pressure is not a variable in HPLC method development, but it is an effect resulting from forcing liquid through a highly restricted flow path. 

Always monitor the HPLC system pressure under all conditions (e.g. analysis, washing columns, equilibration, flushing to waste). When the pressure changes, verify it changes in a predictable manner. Acceptable real-time System pressure depends on: (1) the flow rate; (2) the mobile phase composition; (3) the temperature; (4) the flow path selected (e.g. valve switching, running through the column or to waste). As the column becomes fouled over time, the pressure observed may also change (increase). 

• *COLUMN fouling is one of the most common reasons for the system back pressure to slowly rise over time (usually over weeks) for the same analysis method. Review the sample preparation, injection solvent choice, miscibility/precipitation and/or concentration levels to find the problem.
  

If you change the tubing connections or actuate a valve, (you change the flow-path in doing so), then the pressure observed may also change too. 

Let us consider what other areas of the HPLC system may change the system pressure.

Pump Filters: 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 pump's outlet valve or inside of the prime-purge valve). This filter is designed to collect any piston seal debris or other large particulate contamination from entering the rest of the HPLC system's flow path (i.e. the injector, column, detector...). These small filters (~ 10 to 20um) collect and retain the debris inside the filter so it does contaminate or obstruct the flow path down stream. It is not designed to filter your mobile phase for you (You should have pre-filtered all solutions used in your HPLC). However, this accumulated debris slowly results in a partial obstruction of the flow path, increasing the overall system back-pressure. This may not be obvious to a new user running an analysis method, but the pressure increase due to the clogging filter will occur slowly over time, often masking the change. In a month, it may represent 10-20+ bars increase. In a clean system, if you redirect the flow from the column to waste, you should observe the system back-pressure drop to just a few bars (maybe close to or near zero, depending on the viscosity of the solvent and flow rate). You should know what the "normal" pressure is when the system is directed to waste for many commonly used solvents at typical flow rates. Knowing these values will help you troubleshoot many problems in the future.

• Example: With a new pump outlet filter installed in most 'standard' HPLC pumps, pure ACN solvent directed to waste, running at 1.00 mL/min may show a reading of about 7-bars. If one week or one month later the reading changes to 15-bars, then the filter is clogged with debris and should be replaced. *Perform this type of check on your HPLC pump every day. What is the "normal" back-pressure reading when you direct your typical mobile phase to waste ? What is the value for pure Methanol, ACN, Water, IPA etc. ? It will be different for each HPLC system.
  
• Do you use Aqueous Mobile phase? If so, please filter the final solution through a 0.45 micron (or 0.22u) filter before use. We have observed many laboratories using non-HPLC grade water (e.g. Distilled Water or Sterile Water) resulting in plugging of these pump outlet filters. Always use fresh HPLC grade water (i.e. RO Water) for RP analysis and when preparing buffers.
  

While equilibrating a mobile phase for an analysis the system pressure should stabilize at some point, and also return to the same pressure range after the analysis is complete and the system is allowed to fully equilibrate. As a matter of fact, you should be monitoring the system pressure and detector output after each analysis and wash to determine when the system is ready for the next injection. If the system does not stabilize over a reasonable amount of time, but instead shows a gradual increase in pressure (over the course of minutes, hours or one day), then this may be a sign that their is a partial obstruction inside the HPLC system. While there are many places a partial obstruction could occur (e.g. the injector or column), one of the most common and easy to check for areas is within the pump's outlet filter. Check by diverting the flow to waste and record the system back-pressure. If it is higher than what is expected, the outlet filter should be replaced first. Note: Other problems such as clogged mobile phase solvent pickup-filters or even worn piston seals may also show similar pressure increases too, but most of the time the pump's outlet filter is the cause.

Conclusion: 

• REPLACE the disposable outlet filter found in the HPLC PUMP EVERY MONTH. 

Yes, every single month. These are inexpensive disposable filters designed to protect the flow path of your HPLC system. This is one of the least expensive consumable parts that can have the greatest impact on overall HPLC performance. Stock plenty of these filters and learn how to replace them. Your baselines will be more stable allowing for better quantitation, higher sample through-put, less down-time and less service.

1. For many of the the Agilent 1050, 1100, 1200 and some 1260-series modules using the classic style pump heads, P/N  01018-22707 is suggested ($8.50 USD each). *Please refer to your pump manual to find the correct number for your brand and model of HPLC pump.
   

Syringe Filter Selection for HPLC or LC/MS samples

This article will address the use of disposable female, Luer-compatible, syringe filters without built-in pre-filters for the filtration of individual samples into vials for HPLC or LC/MS analysis. - Note: 96 or 384 multi-well filtering plates provide for a better solution when large quantities of samples need to be filtered. Note: The presented filter membrane material selection criteria also applies to mobile phase filtration too.

The choice of syringe filter depends on the: filter size (volume) of your sample, the chemical compatibility of the housing and membrane and desired pore size. Selection of the wrong filter size can result in too much sample holdup volume (loss of sample on filter) or overloading of the filter (allowing unfiltered material to pass through). If a membrane or housing is chosen which is not chemically compatible with your solution, then contamination of the sample or rupture of the assembly can result. Choosing a filter with too large a pore size can result in material passing through it which could clog or contaminate the solution (i.e. plug an HPLC system or result in a loss of sterility of a solution). Protein binding affinity is another characteristic of filter membranes and if you are filtering samples of biological interest, then you will also want to consider this specification in your selection criteria too (though it will not be discussed in this article).

Syringe Filter Size:

Filters are available in a variety of sizes which are generally in a disc shape and described by their diameter. Common sizes available for chromatography samples include: 3 mm, 4 mm, 13 mm and 25 mm (~25 - 30 mm) diameter discs. The larger the diameter of the disc, the larger the sample capacity, cross sectional surface area and potential hold-up volume of the sample on the filter. 

Hold-up volume is important because some of the sample will be retained inside the membrane and/or filter housing. If too large a filter is selected, samples with small volumes could be lost entirely in the hold-up volume on the membrane. Smaller filters have lower hold-up volumes. To extract as much sample as possible, be sure and use a post-filtration air purge to reduce the total hold-up volume.

If the volume of the sample you wish to filter is under 1 ml, then a 3 mm filter may provide the lowest hold-up volume and require the smallest amount of solution. To filter samples between 1 ml and 10 ml, the 13 mm diameter filter provides a good balance between hold-up volume and large filter surface area. Larger sample volumes from 5 ml to 50 ml are often filtered through the more common 25 mm diameter filters (~4 times the filtration area as a 13 mm disc).

Chemical Compatibility:

Membrane Material: This is where you really must consult the manufacturer’s own documentation for the most compatible filter membrane for both your sample and the solution that will flow through the filter. To simplify the selection criteria, we can make some generalizations about some of the different types available:

Cellulose Acetate (CA): Use with aqueous solutions and a few hydrocarbons only. Low protein binding so good for many biological samples. Not compatible with ACN or DMSO. Can be autoclaved.

Nylon: Great general purpose material and compatible with many HPLC solvents (including THF, alcohols, ACN), but not strong acids. Nylon has a high affinity to bind proteins. Can be autoclaved.

Polysulfone / Polyethersulfone Variants (PS / PES): Commonly used with tissue culture and ion chromatography samples. Stable with many strong bases and alcohols, but few HPLC solvents (as it is hydrophilic). Low backpressure and low protein binding. Not compatible with ACN. Can be autoclaved.

Polypropylene (PP): General purpose hydrophilic material with resistance to most acids, bases, DMF, DMSO and alcohols. Not recommended for use with hydrocarbons, esters or solvents such as ACN. Can be autoclaved.

Polyvinylidene difluoride (PVDF): Hydrophilic material with broad compatibility. Often a good choice for use with alcohols, hydrocarbons, biomolecules, ether and ACN. Low protein binding. Can be autoclaved.

Polytetrafluoroethylene (PTFE): Reported in most brochures to be chemically resistant to almost all solvents, strong acids and bases. Hydrophobic membrane should be pre-wetted when used with aqueous solutions. Low protein binding and very strong. Can be autoclaved.

Most chromatography grade syringe filters are constructed of either HDPE or PP. These materials are compatible with a wide range of HPLC solvents and both offer low levels of extractables. HDPE has been reported to be more chemically compatible with aqueous basic solutions of NH4OH than PP.

Pore Size:

This will depend on your application and a number of different pore sizes are commonly available from vendors (1 micron, 0.8, 0.45 and 0.22 micron are the most common): 

For example, is sterilization of the fluid the goal? If so, a 0.22 micron filter is generally accepted as the best choice.  


For most chromatography or LC-MS applications either a 0.45 or 0.22 micron filters are preferred.

Summary:

• Please refer to the various manufacturers data sheets to select an appropriate syringe filter with: (1) a low hold-up volume; (2) large enough size for the volume of sample; (3) which is chemically compatible with the solution and material you are going to inject through it and (4) lowest protein binding affinity (if applicable).
   
• To reduce the hold-up volume, use a post-filtration air purge to empty the filter.
   
• Minimize contamination from extractables (in the plastic) by pre-rinsing the filter membrane with some of the clean solution. This can reduce the amount of detectable extractables in your sample. PTFE based membranes have some of the lowest extractable levels so consider their use if this is an issue.
   
• If analyte binding is a concern, select one of the membranes which has the lowest binding affinity such as PVDF or PTFE.