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.
   

Speed Up HPLC Analysis Time Using Higher than "Normal" Flow Rates with SMALLER Particles

Column efficiency (as described by Van Deemter) in HPLC is largely a function of dispersion, column particle size and the flow rate of the mobile phase.After a column has been selected, the Flow rate should be optimized for all methods (start with the nominal linear velocity). Once the optimum flow rate range is achieved, little to no advantage in analysis time or solvent savings is found by increasing it (as column efficiency normally decreases at higher flow rates).

From a practical point of view, columns packed with porous 3 to 5 micron diameter supports show only small differences in efficiency as the flow rate is varied above the initial, optimum level (linear velocity). Running at too low a flow rate serves no purpose, increases dispersion/diffusion and delays the peaks from eluting off the column in a timely manner. Higher rates often decrease column efficiency. Once the flow rate has been set within the 'optimized zone', it no longer becomes a variable in HPLC method development. 

Many ~ 3 micron supports do demonstrate some ability to maintain optimum efficiency at slightly higher flow rates (e.g. with linear velocities > 1 mm/second), but significant advantages in using higher flow rates to save time and solvent are not obvious unless the particle size is reduced further. 

With the much smaller diameter ~ 2 micron particles, column efficiency can be further optimized using higher than "normal" flow rates on standard columns. Columns packed with these smaller porous particles show optimized flow rates at much higher linear velocities (e.g. 2x normal or ~ 2 mm/second for standard analytical sized columns, but experiment using 2 to 5x the normal linear velocity to compare results). 

• For example: If your method currently runs at 1.000 mL/min, you may be able to run the same method at 2.000 mL/min OR if your method currently runs at 0.200 mL/min, you may be able to run the same method at 0.400 mL/min or higher using one of the 2.5 or smaller particles. 

This increased efficiency coupled with proper optimization of the HPLC's flow path to reduce dispersion, allows for a doubling of the flow rate without a loss of efficiency (or loss of resolution). Depending on the scaling used, a two-fold savings in analysis time over conventional methods using larger particles may be observed. There may be a corresponding increase in system back-pressure too (* if only the particle size is changed and the column dimensions are unchanged). *Some of this can be countered using proper scaling of the column dimensions too). 

NOTE: Do Not Optimize HPLC Methods for "Pressure". This goes against basic chromatography fundamentals. Back Pressure is a result of pushing mobile phase through the tubing and column and is not a method development tool or variable. As mobile phase composition changes, so does the pressure. Flow rates should be stable. Work within a pressure range that is high enough to permit the pump(s) to function properly, but below the point in which frictional heating interferes with the method.

Optimization of method resolution, overall analysis time and solvent usage should be considered. The increased efficiency gained from the smaller particle size supports also allows for scaling down the column dimensions (i.e. length, ID or both) too, though a trade-off between overall column efficiency vs. analysis time and/or too high a back-pressure must be addressed to optimize the method and meet the application goals.

Summary: HPLC analytical column flow rate is often ignored in method development (* esp after it has been adjusted to the initial optimum, often 1.0 mL/min for a 4.6 mm ID column), but IF you are using porous HPLC particles that are smaller than 3.5 micron diameter, please be sure to investigate if you should re-optimize the flow rate used in your method / application so you can take advantage of any increases in column efficiency and/or scaling. As with ALL applications using these very small particles, pre-optimization of the HPLC flow path is often needed to achieve many of the available benefits.

Backpressure Changes, Pressure Drop from HPLC Tubing Selection (0.007, 0.005, 0.010")

In previous articles we have discussed how the choice of column particle size directly changes the system backpressure. Smaller particles generate higher back-pressures. We have also discussed the importance of HPLC tubing selection to minimize delay volume and diffusion within the HPLC's laminar flow path. Let us now focus on how the tubing's internal diameter and length impacts the total HPLC back-pressure (or pressure drop) observed. 

Key Points:  

1. Try to optimize the plumbing of your HPLC system.  
2. HPLC Tubing lengths between connections (or HPLC modules) should always be as short as possible. 
3. Pressure drop is dependent on the tubing length and inner diameter. Doubling the inner diameter of the tubing will decrease the pressure by a factor of 16.

Once the HPLC tubing connection lengths have been minimized, the next critical dimension which affects band broadening, delay volume and peak-width is the internal diameter (ID) of the tubing. The tubing selected should be narrow enough to reduce the undesirable spread of the peak(s) inside the tubing, but not be so narrow or restricted to result in clogs or obstructions (which is why good chromatography guidelines should be followed insuring that each sample is fully dissolved and filtered before injection). Commonly used tubing ID’s for most analytical HPLC systems are: 0.010” (0.25 mm), 0.007” (0.17 mm) or 0.005” (0.12 mm). By far, 0.007” (0.17 mm) is the most commonly used size for modern analytical HPLC analysis as it offers a compromise between low delay-volume and modest back-pressure (with fewer clogs). However, in addition to the much lower internal volumes which accompany the narrower ID’s, the pressure drop measured across equivalent lengths of tubing may change dramatically and this should be noted during set-up, selection and operation. Take the time to learn what "normal" backpressures are under specified conditions.

 

Understanding how the HPLC system backpressure changes as the internal diameter of the tubing varies is extremely useful in troubleshooting a number of common HPLC problems.

Let us compare the pressure drops measured across three popular HPLC tubing ID’s of the same length (40 cm) using common HPLC mobile phase solvents. This table will help illustrate the observed backpressure changes that the tubing ID and liquid have on the pressure drop.

PRESSURE DROP (in bars):

SS Capillary Tubing, 40 cm length, flow rate 1.000 mL/min.

Mobile Phase / Tubing ID	Water	ACN	MeOH	MeOH/Water (1:1)	IPA
0.010” (0.25 mm)	0.7	0.2	0.4	1.2	1.5
0.007” (0.17 mm)	2.7	1.0	1.6	5.1	6.2
0.005” (0.12 mm)	10.4	4.0	6.3	19.1	24

Note: Pressure drop is also a function of tubing length so if we halve (1/2) the length of tubing used, we also will reduce the pressure drop by one-half. 

Note the four-fold change that narrowing the tubing ID has at each ID reduction. The change is more dramatic when viscous solutions are used (i.e. MeOH/Water or IPA). If you re-plumb any part of your HPLC system with new tubing, then awareness of this physical change will assist you in troubleshooting many types of HPLC problems (to know which types of pressure changes indicate a real problem and which types of pressure changes are normal). Changes to the overall length or ID may result in noticeable changes to the total system backpressure. As an experienced chromatographer knows, when HPLC solvents are mixed together (e.g. gradient analysis) the pressure does NOT always follow a linear progression. In some cases, a reaction occurs between the solutions resulting in an overall change to the final viscosity of the mixture which may not be expected or understood by novice chromatographers (e.g. mixtures of MeOH/Water and ACN/Water are very well know examples which show these properties). 

 

You can download a free, more detailed table of 'HPLC Tubing Backpressure Examples' in PDF Format at this link:

The HPLC Restriction Capillary; Troubleshooting, Qualification and Running Without A Column:

Most types of HPLC pumps will not operate properly without 30 or more bars of back-pressure on their outlets to prevent cavitation and excessive pulsation. Columns play a vital role in stabilizing the baseline during an analysis. In this application, they not only aid retention, but act as a cushion or buffer.

When we want to closely replicate the operation of an HPLC system under "normal" conditions and do not want to use an HPLC column in-line (because a column adds variability), we install a "restrictor" such as a restriction capillary in its place. A restriction capillary is often a very narrow ID section of long tubing (capillary) which will restrict the flow of mobile phase through it. For most HPLC systems, a restrictor which is sized to provide about 1,000 to 2,000 psi (~ 70 to 140 Bars) of back-pressure will closely replicate normal operating conditions. The restrictor can be chosen based on length, ID, volume and your flow rate to create this level of back-pressure. You could place a high pressure rated, zero-dead-volume union its place, but in doing so, the system back-pressure may be extremely low ( a few bars) and show poor pump performance. We need to replicate actual analysis conditions during testing or the results obtained may be invalid and unscientific. An HPLC column, with its densely packed small particles inside acts as a pressure pulse buffer and adds a great deal of back-pressure to the HPLC system. That back-pressure greatly improves the stability of the pump operation and overall baseline. HPLC Columns prevents pulsations by acting as a dampener and/or system buffer.

There will be times when you need to operate the HPLC system without an HPLC column installed.

For Example: 

• Troubleshooting sources of contamination, carryover or artifact peaks on a column;
• Measuring the HPLC system delay volume (gradient delay);
• Testing the performance of the injector;
• Testing the performance of the pump (measure % ripple); 
• Testing the performance of a detector module (measure S/N);
• Running HPLC Operational Qualification Tests (OQ);
• Running HPLC Installation Qualification Tests (IQ);
• Running Performance Verification Tests on a Module (PV);
• Running many of the ASTM Tests (e.g. "Baseline Noise & Drift Test").

Example of a commercially available Restriction Capillary (Agilent P/N G1312-67500). You will want to include any needed details of the restriction capillary chosen for your work in the SOP's that you write which utilize it as part of any test (P/N, source, dimensions, volume...).

UHPLC TIP: Reducing the Column Temperature to Offset Frictional Heating Effects (Causing Poor Resolution)

HPLC column temperature is a critical variable that we adjust and optimize during method development. We use it as a variable during the method development process to improve solubility, optimize peak shape and increase resolution. Once established, it must be carefully controlled during the method analysis to provide reliable and reproducible analysis results. Change the column temperature and you may also change the results obtained. This is a fundamental method development tool and must not be forgotten.

If you are developing a new UHPLC method OR perhaps scaling an HPLC method to utilize 2.5 micron or smaller support particles, then you may observe a loss of resolution or poor peak shape in the new method. There are many reasons why this may occur, and the most common ones relate to not optimizing all of the method parameters correctly when scaling the method (e.g. dwell volume too large, flow cell volume too large, injection volume too large, sample rate too slow, flow rate not optimized, mobile phase composition changes not in scale with the gradient...). But there is another reason...

Resolution may be reduced or lost when all of the initial scaling and instrument set-up parameters are optimized. What is the most likely reason for this? In many cases the use of substantially higher flow rates (relative to linear flow rates) and the use of smaller diameter particles results in much higher backpressures (you may recall that if you halve the particle size, the backpressure increases 4x). The resulting backpressure might be 2, 3 or even 4 times higher than observed in the original method. While these higher backpressures were well within the operating parameters of the HPLC system used, the results obtained were poor. The possible cause? The much higher backpressure increased the amount of frictional heating inside the column, raising the actual analysis method temperature and changing the separation conditions. 

Pushing mobile phase (liquid) through a chromatography column generates heat and pressure. The heat generated increases the actual temperature of the column and reduces the viscosity of the fluid. In conventional columns (i.e. 4.6 x 150 mm, 5u) at 1.00 ml/min, this heating effect is minimal, but at much greater column pressures, > 400 bars, the frictional effects may be substantial. These types of very high pressures may be seen with methods which utilize columns containing the smallest particles (1.9 to 2.5 micron). Enough to change the temperature in the column by several degrees (e.g. >5 degrees C) and result in different method conditions. So, what can you do about this? The most direct way to address the problem is to run the same method at a lower temperature (perhaps decrease by 5 C to start with). This will slightly raise the backpressure (lower temperature equals higher viscosity), but it should cool the column and restore the original temperature conditions used. Additionally, we suggest that you always start column equilibration using a flow ramp to gradually increase the flow over time and reduce the overall heating effect and resulting "shock" placed on the column. An initial delay at equilibration may help reduce these effects (gradually ramp up to the regular flow rate and hold). You may need to try several temperatures and this may be easiest to do if your HPLC has a column compartment with heating and COOLING capabilities. Optimizing the temperature and internal pressures may increase the column lifetime and result in better overall data reproducibility.

HPLC Solvents, Acetonitrile and Methanol, Key Differences and Properties

Widely used in RP HPLC method development, Acetonitrile (ACN) and Methanol (MeOH) are the two most common solvents you will use with water or aqueous buffers to develop methods. So, besides the fact that Acetonitrile is well know to have a higher elution strength / capacity than Methanol [*but NOT at high organic concentrations (e.g. 95% Methanol vs 95% ACN) where Methanol has a higher elution strength than Acetonitrile does], what other properties should chromatographer's be aware of? Let's discuss a few that all chromatographers should know.

PREPARATIONS of MIXTURES (A/B):
First, a few comments about the preparation of mobile phase solutions. 

• Only the purely aqueous portion can be correctly adjusted for pH. Do not try and measure or adjust the pH of the organic or organic solution mixture. 

     There are two common methods of preparing binary mixtures (V/V) of mobile phase solutions.

• Method #1 is to fill a volumetric flask with a specific volume of the "A" solution, then fill the flask up to the line with the "B" solution.

• Method #2 is to fill a graduated cylinder (or volumetric flask) with a specified amount of "A" solution; fill a second graduated cylinder (or volumetric flask) with a specified amount of the "B" solution and then mix the contents of both together.

Whichever method you use, please fully document it in your HPLC method so anyone reading it will be able to accurately reproduce it. The two methods described above are both correct in design, but will result in solutions with different properties.

ABSORBANCE of UV LIGHT:
For HPLC grade solvent (*we should always use HPLC or LC-MS grade solutions in HPLC analysis), ACN has the lowest absorbance (~ 190 nm) of the two making it well suited for low UV applications. HPLC grade MeOH has a slightly higher UV cut-off, around 205-210 nm, limiting its use in the very low UV ranges. *Methods which require low UV wavelengths (<230 nm) should not use Methanol as the primary solvent.

SOLVENT SOLUBILITY:
There is a significant difference between ACN and MeOH in their ability to dissolve many types of buffer salts AND samples. These differences may be critical during method development as higher salt concentrations could lead to plugs, clogs or precipation. 

Solubility of the Mobile Phase:

• A common reason for gradient runs to show poor reproducibility or to fail may be associated with running high concentrations of buffer combined with high concentrations of organic solvent. Most aqueous / organic solutions containing salt solutions of less than 10 mM concentration are not likely to precipitate under most gradient conditions (running to a max of 95% organic, not 100%). If high percentages of organic solvent are mixed with more concentrated buffer solutions, then the higher salt concentrations may precipitate out of solution during the analysis (resulting in clogs, leaks, plugs and/or inaccurate results). Be cautious when mixing organic solvents and buffers together for gradient analysis. Make sure the solutions used will stay in solution and be stable at all concentrations used. Also verify that the buffering capacity is still present when high organic concentrations are used (as your buffer will be diluted). *Not sure if the salt will stay in solution? Just mix up a sample at the same concentration for a test. Look at it. Is there any turbidity or particulate visible? You should have your answer.

• Methanol's overall better solubility characteristics (better than ACN) mean that it often does a better job of dissolving most salts (esp NH4, K and Na) at higher concentrations resulting in better performance and less precipitation.

Solubility of the Samples (changes to Peak Shape, Selectivity & Retention):

• A fundamental requirement of liquid chromatography is that the sample fully dissolves in the mobile phase (initial mobile phase). Dissolve the sample in the mobile phase or in a slightly weaker strength solution (not a stronger solution) before analysis. This insures it will be loaded onto the head of the column as a concentrated slug improving peak shape and RSD. If the sample does not fully dissolve in the mobile phase then you are not in fact analyzing the whole sample. Another area where Methanol may be superior to ACN can be found in its ability to fully dissolve more types of samples. This improved solubility may result in better overall peak shape. Methanol also has different selectivity, often better than ACN (not just the elution strength) which may result in peaks eluting at different retention times than expecting. This is another reason why we always try different mobile phase mixtures containing either ACN or MeOH when developing RP methods. Please never assume that one solvent will be better than the other. Too many novice chromatographer's use only ACN as their main organic solvent for method development. Please don't make their mistake as such a strategy indicates a lack of practical experience and knowledge. You must first try them both separately (ACN & MeOH) to evaluate the results with your own sample (best to start with comprehensive gradients at different pH values, as applicable). You will be rewarded for putting in the initial time to test both types of solutions as no simulator has yet been developed which can predict a truly accurate result with your own sample(s). You may be surprised to learn how many samples show better peak shape and performance using MeOH solutions. If no improvement is seen, document it and move forward with more confidence.

BACKPRESSURE & OUTGASSING:

• ACN is less viscous than MeOH ( 0.34 vs. 0.54 viscosity, respectively) and if used alone will result in lower column and system back-pressures overall. Less gas will dissolve into ACN vs MeOH. Mixtures of ACN and Water will also exhibit an endothermic reaction (cooling the solution) which can trap gas inside the solution. If you pre-mix your mobile phase, let it rest for several minutes after preparation.Mixtures of ACN and Water will show a pressure max around 70% ACN (*This is an unusual characteristic well worth learning).
  

• MeOH is more viscous than ACN alone. It also has an unusual property where a 50/50 mixture of MeOH and Water will result in a much higher system and column back pressure than either MeOH or Water alone will (*ACN has a similar property, but the peak pressure occurs between 60-70%). The effect with methanol is very Gaussian with a peak pressure observed with a 50/50 mixture. An exothermic reaction results from an initial mixture of the two solutions (MeOH and Water) releasing some gas. When preparing solutions it is best to allow the solution to rest for a few minutes to out-gas before topping off or using in the HPLC system.

I hope that this short discussion about some of the differences between these two popular HPLC solvents will aid you in developing better quality HPLC and LC-MS methods.

Reference: Table of HPLC Solvent Properties

Diagnosing & Troubleshooting HPLC Pressure Fluctuation Problems (Unstable Baseline)

Few things in chromatography are more frustrating than dealing with large pressure fluctuations (>1% ripple). If the pump pressure is unstable, and fluctuating up and down, then it will negatively impact your ability to analyze, measure and integrate sample peaks in a reliable manner. A smooth, flat baseline is needed to run and develop methods, collect the data (peaks), integrate and report the results which are reproducible. Baseline instability during an analysis may lead to the entire analysis being declared invalid.

So what causes the HPLC pressure to sometimes fluctuate in a wild manner up and down on your HPLC system? Unfortunately, many things... Most result from poor training, incorrect operation techniques, but some 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 over-time, with the date and list of parts used/replaced. Additionally, maintain a preventative maintenance schedule (e.g. every six months) to inspect and clean the entire HPLC system to check condition, verify operation and minimize unproductive down time. 

HPLC Pump or System Pressure Fluctuation Causes and Solutions:

• Air / gas In the Liquid or Mobile phase (Failure to Degas Mobile phase OR loose fittings) --- Air gets into the system due to a leak or from gas trapped in the mobile phase. Find and correct the cause of the leak and/or degas the mobile phase (use continuous Vacuum degassing or a Helium sparging system only). Leaks are the most common cause of instability, but insufficiently degassed solution is a close second. Make sure your degasser is working 100% correctly (they require professional servicing every 5 years). HPLC pumps require degassed mobile phase for reliable operation.

• Loss of Prime. Improper Priming of the System --- Failure to flush ALL of the lines with freshly degassed mobile phase, before use (every day), will often result in all kinds of instability problems until all of the old gas-filled mobile phase has bee purged from the system. *This could take many column volumes of liquid. Make sure you account for any vacuum chamber volume too. Properly prime the pump heads before use.

• Sticking Check Valve(s) --- If air is exiting the pump outlet, the pump will not function properly. Both Inlet and Outlet valves should be inspected. Remove and clean the check valve(s). Be sure the pump is fully primed with liquid as the check valve might just have an air bubble in it (common on Waters, Thermo and Shimadzu systems). Sometimes sonication of the valve for ten minutes in a beaker containing warm solvent does the trick (e.g. MeOH or IPA/Water). Though very rare, ACN has a bad reputation for polymerizing in solution. If the system has sat unused for a long time OR was not properly flushed out when last used, it is possible that particulate matter may clog the flow path. Small sticky particles may form (ACN polymerization) and cause the check valve to stick inside the housing (use fresh, filtered solvents only to prevent these problems). Clean and inspect any suspect valve first. Replacement of the check valve may be needed in some cases to restore operation. Note, this problem of "sticking" check valves is most likely to be an issue in HPLC pumps with mechanical (gravity or spring) check valves with ruby balls, not modern style active inlet check valves ("AIV") which are electromechanical (solenoid valves) and are very reliable, much less susceptible to these problems. In any case, verify operation of all valves while under pressure (backpressure is needed for them to function correctly).
  

• 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, as this is done at low-pressure). Run a formal pump high-pressure leak test at max pressure to confirm (remove the column and replace with a calibrated backpressure restriction line for all testing). Clean pistons and replace piston seals to repair (you should have spare pistons and seals on hand). *Seals are a maintenance item so expect them to wear out and need regular replacement.

• Flow rate too low (may be 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 pump cavitation, loss of prime, non-optimized piston stroke volume.

• HPLC System 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 (*Water's Article number: 32564 states the back-pressure must be at least 1000 psi for their Alliance systems). Too low a pressure often results in a 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 achieving 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 1% ripple and 0.2% ripple common ("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 one or more of your mobile phase channels may not be 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 (which may dry out).

• 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. Using the wrong value can cause instability.  

• Poor Solubility, Mobile Phase  --- 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 Solvent Pickup Inlet Filters: These can become obstructed or fouled over time (esp. if used with aqueous solutions!). 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). A quick way to check if one filter is causing the pressure to fluctuate is to remove the filter from the one line, then re-test the system. If the problem goes away, then returns when you re-install the filter back on the line, the filter may be obstructed (replace it),

The above list includes some of the most common reasons for unstable baselines. Other non-pump related causes would include a bad / old detector lamp(s) or contaminated mobile phase. To find the cause, test and verify the operation of each component part of the HPLC. Troubleshooting Advice: Test one part at-a-time, before moving to the next part. Never assume anything, test, re-test and verify or prove at each step.