Translator for HPLC HINTS and TIPS for Chromatographers

Saturday, January 25, 2014

Diagnosing & Troubleshooting HPLC Pressure Fluctuation Problems (Unstable Baseline)

Few things in chromatography are more frustrating than dealing with large pressure fluctuations (>1% ripple) when you are just trying to get the HPLC system ready to run some samples. If the pressure is fluctuating a great deal, then it will negatively impact your ability to run samples, measure and integrate them in a reliable manner. A smooth and flat baseline is desirable to accurately develop methods, gather the data (peaks), integrate and report the results. 

So what causes the pressure to sometime fluctuate in a wild manner up and down on your HPLC system? Unfortunately, lots of things... Some are due to poor training/technique, but most are maintenance related so be sure and keep your chromatograph in excellent condition. Maintain a logbook for each instrument and record what types of maintenance and service have been performed along with the date and any parts used/replaced. Additionally, maintain a preventative maintenance schedule (e.g. every six months) to inspect and clean the entire system to minimize unproductive down time. 

HPLC Pump or System Pressure Fluctuation Causes and Solutions:

  • Air In the Liquid --- Solution: Air gets into the system due to a leak or from gas trapped in the mobile phase. Find and correct the leak and/or degas the mobile phase (use a continuous Vacuum degassing or Helium sparging system). Leaks are the most common cause, but insufficient degassing is a close second. Make sure your degasser is working (they require maintenance too).
  • Improper Prime of System --- Failure to flush ALL of the lines with freshly degassed mobile phase before use (every day) will often result in all kinds of instabilities until all of the old gas filled mobile phase has bee purged from the system. *This could take many column volumes of liquid.
  • Sticking Check Valve --- Solution: Remove and clean the check valve. Be sure the pump is fully primed with liquid as the check valve might just have an air bubble in it. Sometimes sonication of the valve for ten minutes in a beaker containing warm solvent does the trick. ACN has a bad reputation for polymerizing in solution. Small sticky particles may form and cause the check valve to stick inside. Replacement of the check valve may be needed in some cases to restore operation. Note, this problem is common to mechanical (gravity or spring) driven check valves, not active inlet check valves ("AIV") which are electromechanical (solenoid valves) and are much less susceptible to these problems.
  • Worn Pump Piston Seals--- Commonly observed as rapid up/down spiking on all channels and an inability to maintain or produce backpressure (the pump will often prime with no problem). Run a formal pump pressure or leak test at max pressure to confirm. Clean pistons and replace piston seals to fix. 
  • Dirty or Restricted Solvent Pickups --- Your mobile phase reservoirs should have pickup filters which are used to introduce the mobile phase to the pump. They keep large particulates out of the system. Over time these filters can clog up or become restricted. Clean or replace them. *A quick way to check if this is what is causing the fluctuation problem is to simply remove the filter from the pickup tubing and see if the flow stabilizes. If it does, then the filters were the cause. Replace them with new ones. Don't run the system without the filters!
  • Flow rate inappropriate for system. Running at a flow rate that is below the optimum range of the specific instrument (i.e. System rated for 200 to 2,000 uL/min, but run at 100 to 200 uL/min or at the limit of the range) may result in an unstable baseline. The cause may be due to cavitation, loss of prime, non-optimized piston stroke volume.
  • HPLC Back-pressure too low to maintain prime in system. Most types of analytical HPLC systems require a minimum system back-pressure of 40 or more bars to maintain enough pressure (mechanical compression) on the component parts to run in a reliable fashion. Too low a pressure often results in loss of prime, cavitation, mixing problems, turbulence and poor reproducibility. Correct sizing of column, particle size, flow rate and mobile phase composition should all take into account acheiving enough back-pressure on the system to maintain a stable baseline throughout the entire analysis. Monitor the system back-pressure at all times for stability. High quality research grade HPLC systems are often capable of maintaining stable isocratic flows with less than 0.2% ripple ("ripple" is a term we often use to describe the pump's pressure output over time relative to the baseline (S/N).
  • Mixing Problem (gradient or isocratic online mixing) --- If your active mixer or proportioning valve (AKA: Gradient valve) is defective or dirty, then only one channel of your mobile phase is getting to the pump. Air would most likely be mixing with the mobile phase causing the unstable flow. Clean or replace the valve. Note: Always try flushing the gradient valve with pure IPA, then DH20 for about twenty minutes. This sometimes restores operation by wetting and flushing the internal seals.
  • Leaking or Worn Pump Piston Seal(s) --- Anything which disrupts the smooth flow of liquid in and out of the pump can reveal itself as a pressure or baseline fluctuation. These are usually cyclical and quickly identified. The pump piston seals are what maintain pressure. If they no longer seal, then they leak. If they leak, then you will have an unstable flow rate. Depending on the size of the leak, it may not be easy to see. To find out if they are leaking, run a full pump pressure test according to the manufacturer's procedure to determine if it can maintain and hold full pressure for several minutes. The test will reveal if the seals are in fact 'sealing'. *Seals are a maintenance item so expect them to wear out and need regular replacement.
  • Wrong Pump Solvent Compressibility Settings --- In HPLC we routinely subject different liquids to very high pressures which can result in measurable liquid compression. The degree of actual compression for each liquid varies, but the modern HPLC pump can compensate for this to improve the accuracy of the mixing and flow delivery.  Most pumps provide for user adjustable solvent compressibility values. If the value input varies a great deal from the actual liquid in the system, then it can result in pressure fluctuations. Example: Water has a value of 46, but Methanol 120 (Search Compressibility on this blog for a table of values). Using the wrong value can cause instability.  
  • Poor Mobile Phase Solubility --- Sometimes the mobile phase which has been prepared (or mixed online) is not 100% soluble. This could be due to an inorganic salt additive which has not gone into solution or failure to fully mix and filter the mobile phase before use. Ultrasonication, a bit of heat and stirring for 20 minutes can help to get everything dissolved. 
  • Dirty inline filter --- A fouled or partially plugged filter can disrupt the normally smooth flow into a turbulent one. Some are installed as part of the pump (i.e. HP/Agilent brand pumps) and should be changed out every month (Yes, for the PTFE frit, replace it once a month with a new one). Other systems use these pre-filters downstream of the pump before the injector. Clean or replace all filters frequently. If used in your system, these are regular maintenance items and should be part of a general 'PM' program.
  • Dirty Mobile Phase Solvent Pickup Filters: Just as with any built-in filter, the multiple solvent inlet pickup filters should be cleaned or replaced on a regular basis to prevent particulate or any material which may contaminate or restrict the flow path from entering the system. Mobile phase pickup filters are often 10 to 20 um and connect to the bottom of the low pressure (e.g. Teflon) solvent lines in each bottle. If you use 316 Stainless steel filter (recommended for organic solvents), they should be removed, cleaned in an ultrasonic bath, rinsed and replaced monthly. If you use sintered glass or other disposable type filters (often used with aqueous solutions), they should be disposed of on a regular basis and replaced with new ones (replacement, not cleaning is recommended because sintered glass can not be sonicated and should be disposed of to prevent bacterial, mold or fungal contamination).
The above list includes some of the most common reasons for unstable baselines. Other non-pump related causes would include a bad detector lamp or contaminated mobile phase. To find the cause, test and verify the operation of each component part of the HPLC. Test one part at a time before moving to the next part. Never assume anything, test, re-test and prove.