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

HPLC Retention Time Drift, Change, Area Variability or Poor Reproducibility. Common Reasons for it.

Retention times and area measurements must be reproducible from run to run. When problems are observed, late, early or variable retention times (and/or peak area values) may be observed. Variation outside of acceptable limits indicates a problem with the sample preparation, method design, function of the HPLC system or a lack of training. Here are several commonly observed reasons why sample (or standard) peak retention times or peak area values may not be reproducible:

(1) TEMPERATURE FLUCTUATIONS:

To obtain reproducible results, the temperature of the HPLC column must be kept constant or controlled during each analysis. Laboratory room temperatures can vary up and down by several degrees during the course of one day and these changes will often change the retention characteristics of the sample(s). The 'On' and 'Off' cycling of power from an air conditioner or heating unit will often cause the baseline to drift in a cyclical manner, up and down, during the day (this can often be seen as a clear sine wave pattern when you zoom-in to study the baseline trace over time). Temperature also changes the refractive index of the mobile phase. Optical (Light) based detectors (i.e. UV/VIS, RI...) will show this change as drift up or down). In some cases, a temperature change of plus or minus one degree C from run-to-run can cause changes in retention times which effect reliability of the method. 

To reduce temperature fluctuations, you must control the temperature of the column and mobile phase (if applicable) during the analysis. This is most commonly done by: (a) using fully equilibrated mobile phase at the start of the day or analysis, (b) keeping the interconnecting lines as short as possible (esp. any which exit the column and go to detectors/flow cells), (c) insulating any stainless steel lines with plastic tubing sleeves to reduce heat loss and (d) using a thermostatted column compartment to maintain the column at a single set temperature throughout the day. Control of the column temperature will remove 'temperature' as a variable from your analysis. Method analysis temperature should be constant from run to run, not a variable. Be sure and document the temperature selected as part of your method. 

(2) INADEQUATE MOBILE PHASE MIXING:

Both high pressure (with separate pumps) and low pressure pumping (one pump with a proportioning valve module) systems depend on efficient mixing to accurately meter the requested mobile phase composition. For gradient analysis, failure to completely mix the mobile phase solution before it enters the HPLC column often results in excessive baseline noise, spikes and poor Retention time reproducibility. If your mobile phase composition changes, then the chromatography will change too (e.g. evaporation of the more volatile organic phase from an open bottle may result in a change in composition). "Mixing" is often accomplished directly in a mixer installed in the flow path of an HPLC pump (For more info, please read this article on selecting a mixer). The associated noise and ripple of incomplete mixing can reduce the limit of detection (LOD) and increase integration error. This mixer is often a static mixer (a simple 'Tee', a tube filled with baffles, a frit or beads, valve orifice or microfluidic device) of low volume design for chromatography use, but allows adequate mixing of the liquids within a prescribed flow rate range. The best mixers incorporate longitudinal and radial mixing in-line. A mixer with too low a volume or of insufficient design can result in poor mixing of the mobile phase (note: incorrect solvent compressibility settings can also cause mixing, flow instability and noise problems too). To reduce mixing problems, first insure that the mobile phases used are fully soluble with each other. Next, make sure that any mixer used is appropriate for the flow rates and volumes you will be using. Monitor the baseline for drift, ripple and artifacts in real time to spot problems and make adjustments to correct them. 

(3) INADEQUATE MOBILE PHASE DEGASSING:

For the best results, continuously degass your mobile phase. Reducing the amount of gas in solution will also improve signal to noise levels of detection, reduce Retention time drift and reduce pump cavitation. If you are using an electronic vacuum degassing module, make sure it is maintained and working 100%. A faulty degasser may cause more damage to your system and methods. Maintain and Repair them just as you do for your other instrument modules. Gas bubbles may cause the inlet or outlet check valves to malfunction (get stuck), baseline noise spikes to appear randomly, flow rates and/or back pressure values to become irregular, detector outputs to show high levels of noise (from air in the flow cell) and also cause the loss of prime or cavitation in pumps. To achieve the best balance of low noise levels and high reliability, both aqueous and organic mobile phases should be fully degassed. This can be accomplished through stand alone vacuum degassing modules or through gentle helium gas sparging. In all cases, degassing must be continuous (not just done one time). Continuous degassing reduces cyclical noise and signal variations. For this reason, I do not recommend using ultrasonic baths to degass mobile phase solutions as these are not used continuously. The mobile phase solution starts to re-absorb gas as soon as you stop sonicating the solution. This results in continuous baseline drift.

Removal of gasses is critical to the function of a modern HPLC pumping system. The liquids used are compressed to very high levels which forces out solubilized gas from the solutions. This is best accomplished before the liquid is transferred into the pump. These gas bubbles must be minimized to achieve desirable baselines. *Even if you use a high pressure pumping system, an inline degassing system reduces the amount of noise and baseline drift. Properly maintain and service your degasser to insure compliant operation. IOW: Always degas your mobile phase solutions.

 

(4) SYSTEM LEAKS or FLOW RATE INSTABILITY:

If the peak retention times have increased over time, one possible reason for this change could be a leak. If your flow rate is reduced by a leak, then the retention times will be longer. Always be alert to this pattern of change and check for any signs of leaks on a regular basis. If you find a leak, do not use the HPLC system until it has been repaired. If there is no leak, then the flow rate may not be what you think it is. 

When the actual flow rate is in question, start by checking it manually (never trust the instrument's display screen or the software value for flow rate. Measure it). An easy way to measure the flow rate involves timing the amount of liquid that exits the HPLC detector line after a defined period of time. For example: If your flow rate is set at 1.000 ml/minute, measure the time it takes to fill a 10ml graduated cylinder. It must take exactly 10.00 minutes.

Inadequate degassing, sticking check valves and/or incorrect solvent compressibility values may also cause flow instability.

(5) COLUMN FOULING: 

One of the most common reasons for changes in retention times or area values of well established peak(s) are due to column contamination and fouling of the support material (or of the inlet frit, guard column). The most common reason for this to happen is due to a lack of column flushing or washing after each analysis (esp when running only isocratic methods). Samples that have been poorly prepared, not filtered or were sourced from a complex matrix (i.e. clinical samples) often contain many compounds which are in-addition to the compound of interest. These materials can be retained on the column and not eluted off during each analysis. They build up over time and cause all kinds of strange problems, including changing retention times, new peaks seen and poor overall or wide peak shapes. 

Gradient analysis provides an opportunity to make sure you use a strong enough mobile phase to elute everything off the column during the run. Make sure you ramp up to a high enough concentration of solvent and use a "hold time" to insure this.

Isocratic analysis is a worst case situation for this to occur as the mobile phase is not ramped up to a strong solvent at the end of the method to push off any late eluters, Instead, they accumulate on the column. 

If you use isocratic methods to analyze samples, then you must follow each analysis run with a second, and separate from your analysis method, "column only wash step". This method does not inject any sample. Instead, it uses a strong wash solution which is compatible with your column AND is well known to dissolve any accumulated material into solution and elute it all off the column. For NP applications an alcohol (e.g. MeOH) may be suitable for this job and for RP applications ACN or even MeCl may be be appropriate. Check with the column manufacturer to find out which wash solutions should be used (do not guess, base it on actual sample solubility). 

(6) SAMPLE OVERLOADING (or too large an Injection volume/concentration for the column): If you inject (load) more sample than the column can hold (as determined by a proper loading study), then the peak that results will often be broader in width with more tailing (from diffusion). This will result in a peak which elutes later than expected, fouls the column and results in poor reproducibility.  Be sure to inject the sample dissolved in the mobile phase (or a solution that is weaker than the mobile phase).

(7) SAMPLE INJECTION VOLUME VARIATION: The injection volume used must be appropriate for the type of injector used. All injectors have a stated range in which they are most accurate. Make sure you are injecting within this ideal range and not at the extreme ends of the range (larger error). Manual injectors with fixed loops should be overfilled (3x) for best results. Autosampler vials must be correctly chosen to be compatible with the injector used, contain an excess of liquid and have a loose cap to prevent evaporation or a vacuum from forming inside the vial. *Test injector accuracy and reproducibility separately, at the volume used for your analysis, as part of your method development review. *Review my article on HPLC injectors for more information.

(8) Changes in the pH OF the MOBILE PHASE: Samples containing ionizable compounds are strongly effected by the pH of your mobile phase. Solutions should be prepared fresh, each day (*acids in solvent may change over time). Buffer capacity is often overlooked (the ability to resist pH change). It is highest at the pKa of the acid/base. Try to work within ±1 pH unit of the buffers pKa value for the best pH control of the mobile phase. If your mobile phase is buffered too far away from its pKa, then poor peak shape or variable retention times are often the result. Note: Weakly ionizable samples can be very sensitive to changes of as little as 0.1 pH unit. 

Common Causes of Baseline Noise in HPLC, UHPLC.

Achieving a flat baseline which does not exhibit spikes, ghost peaks, drift or wander in an unpredictable manner should be a primary goal when performing HPLC analysis or developing methods. Methods which result in flat baselines and have well defined, sharp peaks allow for accurate sample area integration. Integration algorithms perform poorly in quantifying peaks on sloped, drifting or noisy baselines. Excessive baseline noise contributes to many problems, including poor quantitation, high %RSD errors, peak identification errors, retention time variation and many other critical problems. Properly developed HPLC methods are reproducible methods which apply and utilize good chromatography fundamentals. Note: "Noise" is a relative term, often w/o meaning. You should always describe it scientifically, measure and compare the signal to noise ration (S/N) of the baseline vs the peak plus note any cyclical patterns (useful in troubleshooting).

Note: A lack of proper training in the operation of the HPLC system, improper start-up or poor quality maintenance of the chromatograph (Examples: failure to degas and purge the system lines before use; poor mixing; an air bubble stuck in a check valve, a bad detector lamp or a leak will often result in baseline noise) are the main causes of noise. Your HPLC system must be optimized for your specific application. Be sure and allow time for the mobile phase to reach full equilibration with the system before starting any analysis. Do not start an analysis until the baseline is stable.

In this article, we will discuss how temperature fluctuations, inadequate mixing, inadequate degassing and flow cell contamination can result in excessive baseline noise. We will provide suggestions on how to reduce or eliminate these problems. Troubleshooting should be done on-site, not over the web or telephone.

TEMPERATURE FLUCTUATIONS:

To obtain reproducible results, the temperature of the HPLC column must be kept constant during each analysis. Laboratory room temperatures often vary by several degrees during the course of one day and these changes will often change the retention characteristics of the sample(s). The 'On' and 'Off' cycling of power from an air conditioner or heating unit will often cause the baseline to drift in a cyclical manner, up and down, during the day (this can often be seen as a clear sine wave pattern when you zoom-in to study the baseline trace over time). Temperature also changes the refractive index of the mobile phase. Light based detectors (UV/VIS, RI...) will show this change as drift up or down). In some cases, a temperature change of plus or minus one degree C from run-to-run can cause changes in retention times which effect reliability of the method. 

To reduce temperature fluctuations, you must control the temperature of the column and mobile phase (if applicable) during the analysis. This is most commonly done by: (a) using equilibrated mobile phase at the start of the day or analysis, (b) keeping the interconnecting lines as short as possible (esp. any which exit the column and go to detectors/flow cells), (c) insulating any stainless steel lines with plastic tubing to reduce heat loss and (d) using a thermostatted column compartment to maintain the column at a single set temperature throughout the day. Control of the column temperature will remove 'temperature' as a variable from your analysis. Temperature should be a constant run to run, not a variable. Be sure and document the temperature selected as part of your method.

INADEQUATE MOBILE PHASE MIXING:

The associated noise and ripple of incomplete mixing can reduce the limit of detection (LOD) and increase integration error. Both high pressure (with separate pumps) and low pressure pumping (one pump with a multi-channel proportioning valve) systems depend on efficient mixing to reduce noise. For gradient analysis, failure to completely mix the mobile phase solution before it enters the HPLC column often results in excessive baseline noise, spikes and poor reproducibility. "Mixing" is often initially accomplished by combining the flow paths of more than one solvent channel together, using a multi-channel gradient valve or tubing. Mixing also performed directly in a mixer installed in the flow path of an HPLC pump. This mixer is often a static mixer (a simple 'Tee', a tube filled with baffles, a frit or beads, valve orifice or microfluidic device) of low volume design for chromatography use, but allows adequate mixing of the liquids within a prescribed flow rate range. The best mixers incorporate longitudinal and radial mixing in-line. A mixer with too low a volume or of insufficient design can result in poor mixing of the mobile phase (note: incorrect solvent compressibility settings can also cause mixing and noise problems too). To reduce mixing problems, first insure that the mobile phases used are fully soluble with each other. Next, make sure that any mixer used is appropriate for the flow rates and volumes you will be using. If needed, run a gradient valve test to insure that each valve channel is working properly, not leaking or introducing any cross-flow leakage to another channel. Monitor the baseline for pressure stability (% ripple), drift and artifacts  (e.g. spikes) in real time to spot problems and make adjustments to correct them. 

INADEQUATE MOBILE PHASE DEGASSING:

For the best results, continuously degas your mobile phase. Reducing the amount of gas will also improve signal to noise levels of detection, reduce drift and reduce pump cavitation. If you are using an electronic vacuum degassing module, make sure it is maintained and working 100%. A faulty degasser may cause more damage (contamination) to your system and methods. Maintain and Repair them just as you do for your other instrument modules. Gas bubbles may cause check valves to malfunction (get stuck), baseline noise spikes to appear randomly, flow rates and/or pressures to become irregular, detector outputs to show high levels of noise (from air in the flow cell) and also cause the loss of prime or cavitation in pumps. To achieve the best balance of low noise levels and high reliability, both aqueous and organic mobile phases should be fully degassed before and during use. This can be accomplished through stand-alone inline vacuum degassing modules or through gentle continuous helium gas sparging (*Helium makes an excellent choice of gas as it is not soluble in the mobile phase. Never use Nitrogen or Argon gas, they are soluble in the liquid!). In all cases, degassing must be continuous (not just done one time). Continuous degassing reduces cyclical noise and signal variations. For this reason, I do not recommend using ultrasonic baths to degas mobile phase solutions as these are not used in a continuous mode. The mobile phase solution starts to re-absorb gas as soon as you stop sonicating the solution. This results in continuous baseline drift (up and down).

Removal of gasses is critical to the function of a modern HPLC pumping system. The liquids used are compressed to very high levels which forces out solubilized gas from the solutions. This is best accomplished before the liquid is transferred into the pump. These gas bubbles must be minimized to achieve desirable baselines. *Even if you use a high pressure pumping system, an inline degassing system reduces the amount of noise and baseline drift. Properly maintain and service your degasser to insure compliant operation. IOW: Whichever method you use, always degas your mobile phase solutions.

FLOW CELLS:

One other less common cause of baseline spikes and random noise is due to either a dirty flow cell (i.e. the windows) or an air bubble trapped inside the flow cell. If the flow cell is suspected of having one of these problems, then it should be carefully rinsed or flushed out with an appropriate mixture of suitable solutions to expel the air bubble or remove the contamination. If possible, keep a spare, 'known good' flow cell on hand to swap out for troubleshooting purposes. This can help to quickly determine where the problem is. This flow cell must be the exact same size and type (volume and path length) for this purpose. If the cell's windows are contaminated and flushing does not restore them, then many manufacturer's offer kits which allow you to replace the windows and gaskets used. Warning: When attempting to clean or repair any flow cell, be sure and work within the manufacturer's operational specifications for the specific flow cell. Some flow cells are not designed to withstand even very low back pressure and damage can result if you exceed their maximum pressure or chemical rating.

Many other types of problems not mentioned in this short article can also cause baseline noise. For example, a sticking inlet or outlet valve on the pump, worn piston seals, worn out detector lamp(s) or detector electrode (EC) can induce noise. In all cases, the cause must be investigated in a logical, step-wise manner. Demonstrate what is working and rule out items one-by-one.

Reference: http://hplctips.blogspot.com/2014/01/diagnosing-troubleshooting-hplc.html

Gradient Mixing Test For Your HPLC Pump (Step Gradient)

The most popular type of gradient pumping module used to perform HPLC analysis utilizes a low pressure mixing valve in their design. These valves are electronically controlled and proportion the amount of mobile phase from one of several solvent channels into a mixer for introduction to the pump head (*the solenoid valves used for this are sometimes called gradient proportioning valves). They provide random access to multiple solvents (e.g. 4) for method development and column flushing. The mobile phase solutions are mixed at low pressure before entering the high pressure side of the pump head (where they undergo compression). This design requires only one high pressure pumping head and can allow for very high mixing accuracy (often 0.1% per channel) of the mobile phase. This allows for the formation of mobile phase gradients over time which greatly aid in resolving samples apart on the column.

The gradient proportioning valves need to be tested along with the other parts of your HPLC system on a regular basis to insure they are operating within the manufacturer's specifications. They should also be tested anytime you suspect a problem may be present. One quick way to check the operation of two of the valves is to use a tracer compound and STEP gradient to monitor their operation. You can set up a method to perform this test as suggested below.

QUICK GRADIENT COMPOSITION TEST:

Bottle A = 100% DH20;
Bottle B = 0.1 % Acetone in DH20 (*Acetone is the tracer compound);

Flow Rate = 1.000 ml/min;
Column = No column. Install a restriction capillary in place of the column to obtain a backpressure of > 60 Bars;

Detection = 265nm (10 nm bandwidth) UV;

STEP Gradient Program:
    0 to 2.00 min, 0 % B
    2.01 min, 20% B
    4.01 min, 40% B
    6.01 min, 60% B
    8.01 min, 80% B
  10.01 min 100 % B
  12.01 min 20% B
  14.00 min 20% B

Note: If the delay volume (dwell volume) of your system is large, then you may want to adjust the time values shown to LARGER values (i.e. 2 minutes delays are used in this example, but 5 or even 10 minute delays between steps may be more appropriate if your system has > 1 ml dwell volume.

Running the above method should result in a signal trace which shows a step-wise rise to 12.00 minutes (as the acetone concentration increases). The edges of the "steps" should be sharp and the risers should also be close to vertical. The final step change which starts at 10.01 minutes shows a linear gradient change back down to the 20% B level. This line should not have any bumps or dips in it and should transition smoothly back down. The height of the baseline at this point should match the height seen between 2.01 and 4.00 minutes (same 20% B). The height of the proportional steps (e.g. 20, 40, 60, 80) should also be the same. You can use your CDS to measure these height values.

Another useful aspect to view is the S/N ratio at each step. Use your CDS to establish noise windows within each range (e.g. 2.50 to 3.5 minutes). This data is useful when comparing the performance of the pump at different intervals.

If you observe deviations in the height of the proportional steps or dips in the lines, these can be caused by leaking or sticking check valves as well as leaking or sticking gradient proportioning valves. *If you have a quaternary pump, be sure and test all four of the valves used (2x per test).

Lastly, the above example is a generalized method and may or may not be applicable to your specific HPLC pump. Be sure and customize a test method which takes into account the pressure ranges, flow rates, delay volume, mixing volume, and number of low pressure channels used in your pump.

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.