High pressure is a fact of life for HPLC and UHPLC analyses. The force that is necessary to push a liquid mobile phase through a tightly packed bed of tiny particles, even at relatively low flow rates, can cause tremendous back pressure. Fortunately, LC instruments are designed to handle this, but problems will appear when a clog in the system causes the back pressure to climb above the normal range. Clogs arise when particulates are created (in the case of seal wear or mobile phase precipitation) or are introduced (by mobile phase impurities or sample particulates) into the flow path and become lodged somewhere in the various narrow tubing and instrument channels, small-pore frits, or interstitial spaces between column packing materials.
This article will discuss three major sources of these particulates and address some techniques for mitigating them and preventing high back pressure in LC instruments. But effective troubleshooting and establishing an appropriate routine maintenance plan to prevent future problems starts with knowing what “normal” looks like.
What Does “Normal” Look Like?
To figure out if you have a high back pressure problem, you need to know what your pressure normally should be. Having a baseline to compare your actual pressure against can be a tremendous help in identifying a problem and diagnosing it.
Ideally, you would generate a “normal” baseline for the instrument with and without the column installed. By simply removing the column and replacing it with a union, you will see what your normal system pressure looks like. Now, imagine a situation where you’re observing high back pressures in your LC. In that case, replacing the column with the union again and noting whether or not the pressure returns to the normal, system-only pressure will quickly determine if the problem is column or system related. When replacing the column with a union, be sure to use a union that can handle the system pressure (PEEK connectors are reliable up to ≤5000 psi and stainless-steel connectors can be used up to ≤20,000 psi). If your LC software allows you to record your system pressure while you acquire data, that is another great way of keeping records of what “normal” looks like during analysis and column re-equilibration.
Finally, understanding the differences that mobile phase composition has on system pressure can help avoid a false alarm. For instance, switching your organic mobile phase from acetonitrile to methanol will result in an increase in system pressure, all other conditions remaining the same, simply because methanol is more viscous than acetonitrile. Also, during gradient analysis, as your mobile phase composition is changing with the gradient program, pressure will change for the same reason. So, not all pressure changes are a symptom of a problem.
Causes of Abnormally Elevated Pressure
High back pressure in LC instruments is usually caused by foreign material blocking the flow of mobile phase. Although crimped PEEK or stainless-steel tubing will occasionally be the culprit, particulates clogging the system are most often the cause. Note that PEEK tubing is incompatible with tetrahydrofuran and will swell if exposed to it, so avoid that combination to maintain the durability of your PEEK tubing and prevent tubing-related problems.
Particulates are frequently the source of the elevated back pressure, and the following sections cover the main sources of particulates and how to prevent them from clogging your LC system.
Preventing high back pressure in LC systems and protecting your instrument and column against premature maintenance or shortened lifetime starts with your sample. Samples typically contain particulates that can easily clog a variety of components in your instrument. Whether those particulates exist in your sample from the beginning, or they precipitate out at some later stage in the analytical process, understanding the characteristics of your sample and knowing how to mitigate problems it might cause is your first, and one of your best, lines of defence against unexpected instrument downtime for high back pressure problems in LC instruments.
Whenever possible, consider filtering your sample prior to analysis. Syringe filters and filter vials are both viable options for effective filtration. Alternatively, centrifugation is very effective, especially if you pair it with an adjustment of the needle insertion depth to make sure the injector is drawing from the supernatant. Sample preparation to remove particulates can pay dividends when it comes to keeping your instrument up and running, but even if you choose to perform a relatively simple “dilute-and-shoot” technique, having a plan for preventative maintenance can help avoid unexpected interruptions to your workflow.
Even a sample that has undergone extensive sample preparation could pose a risk if the solvent used for the sample is mismatched with the initial mobile phase composition. For example, components of a sample in DMSO may end up crashing out of solution immediately if introduced into a highly aqueous mobile phase, and that situation can cause clogs, especially in the column. This mismatch can also result in poor chromatography, especially for early eluting compounds.
Using a guard column or an UltraShield pre-column filter and changing it as part of your routine preventative maintenance plan, can mitigate the risk sample particulates pose to your analytical column. If something is going to clog, it is best that it is not your analytical column! When using a guard column, always select the system that is designed for your analytical columns. Restek offers three guard column systems: EXP guards for Raptor and Force columns; Roc guards for Roc columns; and Trident guards for Ultra, Viva, and Pinnacle DB columns.
The Mobile Phase
The two main contributors to system or column clogging due to the mobile phase are bacterial growth in poorly maintained aqueous mobile phase bottles and buffer salts that precipitate out of solution, typically as a result of large changes in mobile phase composition. In all cases, be sure to only use HPLC-grade chemicals in the preparation of your mobile phases. And it’s always a good idea to use mobile phase filters, which are available in both glass and stainless steel.
The best way to keep your aqueous mobile phases free of bacterial growth is to make sure they are made fresh and kept capped. High throughput labs are not likely to run into this problem because their mobile phases will be used and replaced long before they have a chance to grow bacteria, but a week or two of storage, especially at mid-range pH values, is an invitation to grow system-clogging bacteria. However, even in high-throughput cases, any residual mobile phase should be discarded, and the bottles cleaned between uses to further mitigate the growth of bacteria. Another remedy is to use opaque or amber solvent bottles to block out the light needed for microbes to grow.
Even bacteria-free mobile phases can cause high back pressure in LC systems if their buffering salts precipitate out of solution as a result of gradients that push mobile phase composition to the point of insolubility. For instance, a gradient that moves to a highly organic phase, especially when using acetonitrile, may result in buffering salts precipitating out of solution and creating obstructions in the chromatographic system. Or, switching lines in your LC without properly flushing the system out could result in highly organic mobile phase moving through lines that previously contained buffered mobile phase, resulting in salts precipitating in the pump.
Instrument Wear and Tear
There aren’t too many places where instrument wear and tear will result in high back pressure problems, but they do exist. Pump seals eventually wear down, and that wear can be accelerated when buffers are used. Particulates from chewed-up pump seals can pose a risk, so replacing the seals and any inline filters meant to stop particles generated from them should definitely be a part of your routine maintenance schedule. Additionally, as the first point of contact with your sample, it’s not uncommon for your needle or needle seat to become clogged. Also, normal wear on an auto-injector rotor can result in pieces being shed from the rotor material, which can then clog the channels or make their way further downstream. You can avoid having these issues surprise you by having a preventative maintenance schedule in place that routinely replaces these parts. To simplify this, Restek offers convenient preventative maintenance kits that include everything you need for logical, comprehensive routine maintenance of high-wear and problematic components. Individual parts such as piston seals, rotors, and needle seats are also available for unplanned maintenance events.
Troubleshooting Elevated Pressure
Even extensive sample preparation and a good routine maintenance schedule won’t completely eliminate the possibility that you will encounter abnormally high back pressure in your LC instrument. Knowing what “normal” looks like will help you identify problems when they arise, but to accurately diagnose where the system is blocked it’s best to isolate one potential source at a time. Starting at the detector and working backwards up the flow path using a systematic approach is the best way to locate an elevated pressure situation and determine the root cause. By either adding or removing components one at a time, you can easily identify where the problem is and resolve the issue causing elevated back pressure. In all cases, as you troubleshoot and work to pinpoint the problem, be sure not to expose your analytical column to unnecessary high-pressure cycles or else you may damage your column in pursuit of the clog. As a last step, be sure to document of all routine and nonroutine maintenance events: a quick review of instrument records can help you determine if future problems can be prevented by adjusting your routine maintenance plan.