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.
   

Agilent Quaternary Pump (e.g. G1311A ) "Secret" Operator Tip to FLUSH the HPLC Pump in 1/2 the time!

One of the most popular "tips" taught in our Agilent 1100 and 1200-series HPLC training classes shows users how to speed up the daily priming and flushing process of the Quaternary Pump. Many people use these pumps without taking advantage of the Quaternary pump's higher flow capability. If you are not currently using the higher 10mL/min flow rate capability offered by this pump (vs. the Binary pump's 5 mL/min), then you are missing out on a free time saving feature. Please read on to learn how to use this feature.

Based on the HP 1050 pump and introduced in 1995 as the "1100-series" version, the G1311  "Quat" pumps are one of the most popular research grade HPLC pumps found in laboratories today. They are extremely reliable, rugged, easy to operate and service. The Quat pump is driven by an easily accessible, single pump head with an in-series, servo controlled dual plunger and Multi-channel Gradient Valve ('MCGV') for 4-channel solvent proportioning with an active inlet valve (known as the 'AIV', first used in the HP 1050 pump and the reason for this pump's high reliability. No more "sticking" inlet valve issues!). Unlike the Agilent Binary pump (G1312), which uses two separate dual plunger pumps (2-channel) at up to 5.0 mL/min (maximum), the Quat pump offers an extended flow range, up to 10.0 mL/min (maximum). However, most users are not aware of this or do not know how to utilize this higher flow rate feature because the Quat pump defaults to a maximum flow rate of 5 mL/min at initialization. The ability to program the pump to operate at flow rates greater than 5 mL/min requires a "trick" to activate it (which apparently is a secret as we rarely encounter customers who are aware of how to use it). 

Let me share with you why you would want to use this feature, why the feature is hidden to most and of course HOW TO ACTIVATE IT on the Quat pump.

• Q: Why would you want to run the pump at 5 to 10 mL/min? Semi-prep columns can be run within this flow rate range, but a more common reason to operate at 10 mL/min is for daily system start-up. Anytime you replace or change the mobile phase bottle/solution OR when you startup the HPLC system (each day) one of the very first things you need to do is prime or flush each of the mobile phase channels, one-at-a-time through the system to waste. Air bleeds into the system when it is not used and this procedure primes the lines and pump head with fresh mobile phase preparing it for use. The system's flow path is directed to waste (via the open, prime-purge valve) during this step so back-pressure is not a concern. The higher the flow rate you can use for this flushing step, the sooner you can complete it. If you run the pump at 10 mL/min vs 5 mL/min, then flushing can be completed in half the time. This is especially useful if you have a model G1322A degasser module installed as the internal volume of each degassing channel in the G1322A is 10-12 mLs, requiring extended flushing times (4x channels = 30+ mLs flush per channel) before moving on to the next channel.

• Q: Why does the Quat pump initialize with a reduced, 5 mL/min maximum flow rate? The Quat pump was designed to meet two different operating pressure ranges. From 0 to 5 mL/min the permitted operating pressure range is 0 - 40 MPa (0 - 400 bar). Above 5 mL/min, the operating pressure range is reduced, 0 - 20 MPa (0 - 200 bar). As most analytical chromatography is performed at flow rates below 5 mL/min, the system initializes using the more practical, 0 - 400 bar range, limiting flow rates to 5 mL/min maximum. The default maximum pressure field is set to 400 bars. You should always change the maximum pressure value from 400 bars to a more realistic maximum pressure (lower value) for your method. Use a maximum value that is appropriate for your own method. *The only time you will want to set it to the maximum value is when conducting a Pump Pressure/Leak test (it must be set to max pressure for testing).
  

• Q: When I try and enter a pump flow rate larger than 5 mL/min, the system does not accept it. How do I program the pump to increase the flow rate past 5 mL to 10 mL/min? In order for the system to accept a flow rate of greater than 5 mL/min, you must FIRST set the maximum pressure limit to a value that is 200 bars or less (within the allowed "0 - 20 MPa (0 - 200 bar)" range). Once the maximum pressure limit has been reduced in the method, the system will then allow you to enter a higher flow rate such as 9.999 mL/min (10 mL/min). As long as the maximum pressure alarm is set within this window (200 or less), the pump will allow flow rates above 5 mL/min to be used. Now you can program the pump to flush lines or prime the system at twice the speed of the Binary pump equipped systems (10 mL/min).

Please share this "trick" with other users of the G1311A, G1311B, G1311C versions of this pump so they can maximize their time and productivity. Let us know if you find this tip useful.

Terminology. Which is it? "UPLC" (TM) , UHPLC or HPLC? The correct name is still HPLC.

Proper terminology is very important in science. Brand names or trademarks should not be confused with the names of techniques or methods. I sometimes hear and see people misuse the terms "UPLC^TM " and/or "UHPLC" so think we need a short post to clarify the correct use of these terms. Here are some basic definitions of the terms plus background.

"LC" or Liquid Chromatography. A general name for any type of chromatography where liquid is used as a the carrier phase and a solid support is used as the media, often packed into a tube.

 "LPLC" or  Low Pressure Liquid Chromatography. LPLC often includes chromatography analysis using glass or plastic columns with very large particle support beads run at pressures ranging from atmospheric (gravity driven) to several hundred psi (~ 30 bars max, but more commonly just a few bars). Very large particles are required in this application to aid flow through the support, which in some cases is gravity driven, but in others, a small pump is required to push the mobile phase through the column.

"HPLC" or High Performance Liquid Chromatography: *Used to be called High Pressure Liquid Chromatography to differentiate it from the previous term "LPLC". Now we refer to it as "high performance" chromatography, though both terms are technically correct. Compared to the very large media used in LPLC (mm), HPLC uses micron sized support particles packed under very high pressures in stainless steel (note: sometimes strong rigid plastic columns are used for biocompatible applications) columns to enhance the resolution obtained by many orders of magnitude. As such, the more popular definition changed from "High Pressure" to "High Performance" Liquid Chromatography to emphasize this improvement, but once again the technique itself did not change (marketing). Today, we still refer to all modes of high pressure liquid chromatography separation techniques as "HPLC" or sometimes just "LC". The most commonly used HPLC pumps are rated between 400 and 600 bars maximum pressure (with some capable of 1,200+ bars) though in normal use, we rarely run methods which use pressures over 300 bars.

"UHPLC" or Ultra-High Pressure Liquid Chromatography (or Ultra High Performance Liquid Chromatography) has become both a new marketing term and perhaps a subcategory of HPLC (other subcategory examples include: nano-HPLC, narrow-bore HPLC, and mini-bore HPLC). UHPLC is presently defined as the use of sub-2 μm particles with a low dispersion HPLC system, optionally with a pump capable of > 600 bars pressure. UHPLC is still HPLC. Nothing changed except it implies you will use sub 2-micron particles in the column (in other words, just the particle size is highlighted). Many methods which use sub-2 μm particle columns can and are run on a low-dispersion HPLC systems at pressures which do not exceed 400 or 600 bars [For more information, please read: "Pressure Drop Across an HPLC / UHPLC Column"]. The technique used in all cases is still correctly called HPLC and should be described as such in papers, articles etc. 

• For example: We have been using narrow bore columns (2.1 mm ID) containing small particle supports for more than 30 years and never changed the name of the technique used each time we changed the column support type used (e.g. 20u, 10u, 5u, 3.5u, 2,5u, 2.2u...). As a matter of fact, in the late 1980's and early 1990's there was a bug push to use 1.0, 2.1 and 3.5 mm ID columns with 3.5u and smaller particles on low dispersion systems to both save solvent, increase performance and reduce run times. This required the use of HPLC systems which were optimized with low dispersion flow paths such as the Hewlett-Packard model 1090 HPLC system (DR5 pumps). Perhaps the technology and methods came too early? Columns with very small particles proved difficult to pack (poor RSD batch to batch). The solvent savings and reduced run times just did not interest people at that time and after a few years, the lack of interest resulted in few commercial columns being available with these properties (I recall packing many of them myself in the lab).

"UPLC^TM" or Ultra-Performance Liquid Chromatography is a Trademark of the Waters Corporation. Waters Corporation uses it as a marketing term for their own product technology. Defined by Waters as, "the use of sub-2 μm particle columns in combination with low dispersion, high pressure (15,000 psi or 1,034 bar) instrumentation". The confusion seems to come from: (1) New users of HPLC who think that the name "UPLC^" is the name of the technique OR (2) HPLC user unfamiliar with other brands of instruments, using the Water's trademark of "UPLC" to describe the technique of HPLC or an HPLCinstrument with a pump which is capable of exceeding 600 bars pressure OR having a low dispersion flow path. They should be using the term HPLC in all of these cases or optionally, UHPLC, another general marketing term, not "UPLC" (unless they really are referring to a Waters product name or specifically, technology).

Summary: In general, as long as the back pressure is above ~30 bars and/or you are using packed LC columns with particles less than ~ 50 microns in diameter (newer, monolithic supports and superficially porous particles also qualify), then the technique used is always called HPLC. If you are using sub-2μm particles and the system operating pressures for the method are at or above 600 bars, then the term "UHPLC" could be used as well (not UPLC^® unless you are specifically using a Waters Corp "UPLC" branded system under the same conditions described), but the term HPLC is far more accurate. You are always correct describing the techniques used as HPLC and we encourage you do so in all articles, papers and discussions.Key take away ... Changing the particle size of the support used AND/OR operating with system back-pressures above 600 bars does not change the name of the technique used (It is still "HPLC"). Please do not confuse marketing names (created in the hope of selling more systems) with the actual name of the analytical technique.

Appropriate Mixer Volume for HPLC and UHPLC Applications

For gradient analysis, most analytical scale HPLC (UHPLC) systems incorporate a solvent mixer which is designed to balance the requirements of moderate dwell volume, low noise and good mixing efficiency. Depending on the method run, the ideal mixer's volume may in fact be completely different than the one installed in your chromatography system. A high-pressure mixing Binary pump can often work well with a slightly lower volume mixer than a low-pressure mixing ternary or quaternary pumping system (because the high pressure mixing gives you a head start), but both pump types benefit from additional mixing.

• Be sure to also consider the volume of any pulse dampener used too as these often have large internal volumes and act as mixers. Some pulse dampeners also incorporate the pressure transducer and/or mixer. These types of combination modules may limit the types of modifications which can be made to optimize the mixing and reduce the dwell volume.

• Don't forget to address the dwell volume contribution of the autosampler, injector loop, interconnecting tubing (extra column volume) and detector flow cell too when optimizing the flow path of your HPLC system.

Here are some general guidelines to help you determine the appropriate mixer volume for your own HPLC system. Note: Since many types of mixer designs exist (static, dynamic, shear...), these are guidelines only. There are some commercially available, high efficiency, low-volume mixers available which can reduce the need for a large volume mixer. Your specific application should be taken into account to determine which size is best.

HPLC System Mixer Volume Choices - Size Matters ("Mixer Volume")

SMALL: Fast or ultrahigh speed separations using low volume, small particle columns. These types of applications depend on a low dwell volume mixer for gradient analysis. To achieve this, your HPLC system should be plumbed with narrow bore capillary tubing (example: 0.005" ID; 0.12mm ID) and include a gradient mixer with a volume of less than 100 ul for low flow rates (example: ~35 ul is rather common size). 

LARGE: High Sensitivity Analysis: Gradient analysis where sensitivity is key, benefit from larger volume mixers to minimize contributions of any UV absorbing additives (e.g. TFA) and turbulence in the flow. Traditional 300 to 750 ul mixers often work well in these applications, provided that the column volumes are also large. Smaller column volumes will require smaller mixer volumes to reduce the added dwell effect.

MEDIUM: Routine HPLC Analysis: Typical analytical separations using 3 to 5 mm ID columns (x 100 mm or longer) usually benefit from modest sized mixers within a range of 200 to 400 ul volume. For these applications, I often start with a recommendation to use a mixer which has 10% of the columns volume as a starting point. For a typical 4.6 x 250 mm, 5 micron porous support column, which has about 3 mLs of internal volume, a 300 ul volume mixer usually provides enough mixing volume for routine gradient analysis.  


Additional Info:

Back in the 1980's we often related mixer volume to intended flow rate/column dimensions. For example: A mixer size of 25 ul was suggested for 50 ul/min flow rates (commonly used with 1 mm ID columns). A mixer size of 200 ul was suggested for 200 ul/min flow rates (commonly used with 2.1 mm ID columns) and 350 ul mixer volume for 1.000 ml/min flow rates (commonly used with 4.6 mm ID columns). Note: Mixers such as these, with large volumes relative to the column volume contributed to large gradient delay times, but this was, and still is, of less concern for isocratic methods.

As mentioned before, the type of mixer, column volume, flow rate and mobile phase characteristics will help suggest the most applicable volume for your application. When in doubt, select a larger mixer volume for isocratic analysis (less baseline noise, better for gradients) and a smaller one if reducing gradient analysis delay volume is critical.