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HPLC Troubleshooting Guide to Cycling Baselines and Pressure Fluctuations

The Problem

A short term cycling baseline is seen along with pressure fluctuations (Figure 1).

Figure 1: Short term cycling baseline.

This page is part of our series of 6 free Chromatography Troubleshooting Guides.

HPLC

Changes in Selectivity, Retention
and Resolution »

Cycling Baselines and Pressure Fluctuations »

Solving HPLC Peak Splitting Problems »

GC

Broad Peaks and Loss of MS Sensitivity »

Broad, Double/Split, Fronting Peaks »

Peak Area Reproducibility and Reduction
of Sensitivity »

 

The CHROMacademy HPLC Troubleshooter

Both chromatographic (up to three) and instrument symptoms (up to two) can be selected at one time.  The chromatographic symptom which is being seen is a short term cycling baseline along with pressure fluctuations, which is instrument related (Figure 2).  Each option has a helpful rollover which illustrates the problem; this can be useful if you are not entirely sure what problem you are seeing.

 

Figure 2: Input of chromatographic (top) and instrument (bottom) symptoms into the CHROMacademy HPLC Troubleshooter.

 

As if by magic!  In three simple steps we can now access possible answers to our problem (Figure 3). 

The Troubleshooter has provided 28 possible causes of our problem.  Troubleshooting is inherently complex, especially when you have multiple symptoms, however, even with 28 possible causes there is no need to panic and feel overwhelmed. 

The possible causes of the problem are rated using a unique, proprietary star system based on the number of symptoms which are matched and the likelihood that your particular combination of symptoms is caused by that particular problem. 

They are then ordered by the likelihood of that cause having resulted in the problem being investigated, which can save you a huge amount of time in investigating improbable causes.

One of the golden rules of troubleshooting is try one thing at a time. 

This means that we can start at the top of the possible causes list and work our way through each option until our problem is resolved. 

  try our hplc troubleshooter

 

Figure 3: CHROMacademy HPLC Troubleshooter results page, including causes of cycling baseline and pressure fluctuations,
solutions to each individual cause, and further resources for resolving the problem.

 

Another great troubleshooting mantra that should be kept in mind is “use some common sense”.  For example, if a cause is suggested that is highly unlikely, i.e. a problem with a refractive Index detector when you are using UV, this is unlikely to be the answer you are looking for.

By clicking on the suggested cause, useful information is displayed in the solution window to instruct you how to remedy the problem and also measures which can be taken to avoid the problem in the future.  Finally, there are further resources listed for extra reading to help increase your troubleshooting abilities.  These resources come from the CHROMacademy multi-media learning database, LCGC magazine, and our many instrument vendor partners and there are over 1 million separate resources for you to learn from.


For some golden rules of troubleshooting see the following CHROMacademy pages:

Chromatography Troubleshooting Tips »
 

The Solution

Troubleshooter results in hand we were able to begin to try and resolve the problem of the cycling baseline and pressure fluctuations.
In an ideal world the first troubleshooter result would be the correct one, however, troubleshooting is often not that kind.  In this case we had to work through the first five possible causes (not an outlandish number of tasks to be carried out).  We primed the pumps, made fresh eluent and ensured it was degassed, calibrated our pump flow rate, and checked for leaks at all fittings.

  1. Air in pump (cavitation in one or both pump heads)
  2. Air in mobile phase
  3. Pump flow rate varying (longer term variation as opposed to pulsations)
  4. Leaking pump
  5. Leaking piston seal

It was found that there was a small leak coming from the bottom of the pump head, which was attributed to a perished piston seal.  Piston seals are designed to deform around the piston, and as such, wear and tear and the need for replacement is expected.  When the pump head has been opened for this type of maintenance it is always advisable to check the piston for scratches and pitting.  Pistons can be cleaned with solvent and even mildly abrasive pastes. 
The piston seals were replaced and the instrument was started, however, much to our dismay the baseline was still showing short term cycling.

Figure 4: Short term cycling baseline following replacement of a leaking piston seal.

 

However, do not panic.  It had been detailed in the troubleshooter solution that the piston seals required softening in order to deform properly around the piston and create a tight seal.  Most manufacturers recommend a wear-in procedure after the installation of new seals. 

This consists of flushing a solvent through the system under relatively high pressure for 40 minutes to 2 hours.  Propan-2-ol is a good solvent to use as not only does it soften the seals to help them deform, but it also increases the backpressure due to its high viscosity.  A long length of restriction capillary (narrow I.D.) or an old ‘dead’ column can be used to help increase the pressure to typically over 200 bar.

Following this operation we were very happy to see that the baseline had returned to normal (Figure 5).

Figure 5: ‘Good’ HPLC following replacement of piston seal and proper wear-in process.


Some helpful hints to avoid cycling baselines are:

  • Avoid air in the system by making sure that all mobile phases are properly degassed using an appropriate method (even when using in-line mobile phase degassers).
  • Always purge the pump prior to operation.
  • If your system has a vacuum degasser, ensure all connection are tight and not susceptible to ingress of air.
  • Implement a pump flow check after major maintenance.
  • Check for leaks regularly.

For even more great troubleshooting tips watch the following webcast:

HPLC Troubleshooting - Eluents and Solvent Delivery Systems »

 

Have you ever had a problem with the following symptoms?

Variable peak height/area

 

Negative baseline drift

Use the CHROMacademy HPLC Troubleshooter to see what could be causing the problem and how to resolve it.

HPLC troubleshooting

 
 

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