HPLC SOLVENT COMPRESSIBILITY - REVISITED

 Twelve years ago I published a short article here (HPLC PUMP SOLVENT COMPRESSIBILITY VALUES) which described the importance of setting the correct solvent compressibility values in the HPLC pump's table. Developing HPLC methods which exhibit smooth, stable baselines, with little measurable signal artifacts (e.g. spikes, noise, oscillation) and minimal pressure fluctuations help insure reliable, repeatable methods. Taking steps to insure that the LC pump operates is setup properly for the method are part of following good chromatography fundamentals

Over the past month I consulted for three different clients who needed help in troubleshooting various "pump stability problems". In all three cases, each HPLC system showed extreme pump pressure cycling, cavitation, noise and instability over time. Pressure fluctuations of 10% (or in one case, 10-30% Ripple values) were observed in several different HPLC methods that were used. One of the very first areas to check for problems with pump pressure instability is mobile phase degassing.  

Proper operation of the HPLC pump requires that efficient degassing of all mobile phases is performed before the liquids enter the pump head. 

Failure to properly degas liquids often results in pump cavitation, check valve sticking and baseline instability. An Inline vacuum degasser or continuous Helium sparing should be used to degas all mobile phase solution for use in HPLC (not sonication or vacuum filtration which perform poorly to solve degassing issues). 

In one of the three cases, the HPLC degasser was found to be broken and long overdue for service. Cleaning and servicing the degasser cured the problem and the method that once showed pressure ripple of >10% now shows no baseline disturbances and very low ripple of ~0.1% at ~ 70 bars system pressure. 

Before I was called in to assist each client, the clients had replaced numerous parts, including: pump seals, check valves, mixers, solvent frits and still had the same baseline instability issues (no change). As recommended by me, two of the clients had their very old degassers cleaned and serviced (as they were long overdue for service), but still had some baseline and pump instability (servicing the degassers improved the baselines, but the pump was not running as it should). In both cases, the cause for the remaining pump instability was quickly identified by me on-site (many problems can be quickly diagnosed on-site).

• The client had incompatible solvent compressibility values stored as part of their HPLC methods. This resulted in huge baseline disturbances, spikes, cavitation and occasional loss of prime. 

One of the clients normally ran methods containing high percentages of ACN (with some water) for their sample methods, but a few months earlier had switched to running with gradients containing high percentages of methanol. The solvent compressibility values stored in their system were appropriate for WATER, but they never updated them when they used the same method file to run samples in mostly methanol solutions (which need different compressibility values). Though they all had been using HPLC for many years, they had not received basic HPLC instrument training to know how to adjust and optimize these and other important instrument settings for EACH method (they were overwriting each new method, a common new user mistake, when making changes). Once we changed the method's solvent compressibility value to a more compatible one (in their case, for methanol), the baseline smoothed out in just a few minutes and all of the pressure instability issues went away (*they had replaced several thousand dollars worth of perfectly functioning parts trying to solve this issue before I arrived). Professional training in how to use and operate any HPLC instrument should always include how to set and optimize the compressibility value(s). Make sure you know how to incorporate the correct value in each new method that you create. Always spend up-front time to optimize each method for the application before you use it to analyze real samples. The initial time spent getting everything to run smoothly and reliably will improve overall accuracy plus save money and time.

• Note: In a low-pressure HPLC single-head pumping system with multi-position solvent selector valve (e.g. Most ternary or quaternary systems) one value is allowed, but in a true, dual-head binary pumping system each of the two pump-heads may have a separate field to input the solvent compressibility values.

The importance of inputting the correct and applicable solvent compressibility value(s) into the pump's settings, for each solvent used is one of many steps in creating an optimized HPLC method. There are no universal values, but the instrument manufacturer will have included a generic value in the pump's compressibility settings field. Should you use this generic value?  What are the chances that a randomly selected value used as a 'place holder' in the software is the correct value for your method?  Just as with flow rate, solvent composition, run time, stroke volume, wavelength etc., entering (and saving) the correct solvent compressibility value into EACH method helps to optimize the pumping performance. You will want to select an appropriate value FOR EACH AND EVERY HPLC METHOD YOU CREATE and use (and be sure to save the method with a unique name). Start by loading your HPLC method into the system, then look at the solvent compressibility value(s) used. Are they correct? Change the value(s) shown to values that are appropriate for your method. It is OK to experiment and try different values (we encourage it!). Monitor the S/N levels of the baseline noise for comparison. The instrument manufacturer should provide a table of suggestion solvent compressibility values for use with their system [For HP/Agilent systems, you can see an example table at the link I provided in the first paragraph of this article or review the operator's manual for more information].

• A related article to this one that you may find useful is; "Diagnosing & Troubleshooting HPLC Pressure Fluctuation Problems (Unstable Baseline)";
  

HPLC Injection Volume: What Should I Dilute It In and How Much Sample Can I Inject?

HPLC Injection Volume and Solution Tips: For best results, the choice of injection solution and amount must be carefully selected. Successful HPLC & LC-MS methods shall observe good chromatography fundamentals. 

• How much sample can I inject on my column? The HPLC injection volume must be carefully selected to avoid overloading the column and also maintain good quality peak shape (Good peak shapes, Gaussian are ideal, are preferred for accurate integration and quantitation). Too large an injection volume and the peak shape may be broad and result in co-elution, column fouling and/or poor reproducibility. Too low an injection volume may lead to no-detection, poor reproducibility and/or inaccurate integration. Choose an appropriate Injection Volume (and concentration) that is appropriate for the COLUMN and Method used (their is no universal answer as they depend on YOUR column and method). Start, by learning what your HPLC column's "dead" volume is (Determining the HPLC Column Volume Link here).  As a general guideline, keep the volume low and inject no more than ~ 1% of the column's dead volume (maximum for most columns is ~ 1 to 2 %, but if the peak shape is excellent, sometimes up to 3% is possible). The actual capacity will be different for different column support types, dimensions etc, so it is best not to guess. Use a volume that is within the injector's most accurate range (for most auto-injector's, the optimal range may be found away from the extreme limits, often between 20% and 80% of capacity, but please refer to the documentation for your injector for specifics). Once an acceptable volume has been identified, then you can vary the concentration to find the best sample load for your analysis conditions.
  
• NOTE: To find the true and correct answer to "How Much Can I Inject (Load) onto my column" requires that you conduct a 'Loading Study' [To run a loading study you will prepare a batch of samples of increasing concentrations levels which can be individually injected, then evaluated on YOUR column, using YOUR method. This is how we determine the MAXIMUM amount possible which can be loaded and still provide good quality results. All other methods are just estimates.

• What should I dilute my sample in? Dilute samples using the mobile phase solution (in the case of gradient compositions, use the "initial" mixture to avoid precipitation). Your sample(s) should be FULLY dissolved in the mobile phase and not in a solution that is chemically incompatible with the flow path or is "stronger" in elution strength than the initial mobile phase. The diluent should not interfere with the analysis or loading of the sample onto the column. Example: If your method is 100% aqueous, then do not inject the sample(s) in a solution that contains organic solvent (i.e. ACN). *Peak fronting, splitting, precipitation and/or distortion (broad shapes) may result from using a diluent that is stronger than the mobile phase.
  

• My sample solution is cloudy or has "stuff" floating in it. ONLY Inject sample solutions which are 100% fully dissolved, in-solution. Injecting samples which have precipitated out of the solution OR which are not fully dissolved in solution (100%) may result in line obstruction, clogging, column fouling and invalid data collection/results. Take the time to find a mobile phase that your sample fully dissolves in to avoid problems. Troubleshooting and repairing an HPLC system for clogs and/or column contamination is both time consuming and expensive.

• Filter sample solutions to prevent clogs and reduce column fouling. Make sure the sample is first fully dissolved in the solution and do not use a 'filtering' step as a cheat to remove undissolved sample. Filtering is used to protect the system from particles that we can not easily observe which may clog the system. Please refer to the article; "Syringe Filter Selection for HPLC or LC/MS samples"; for more information on filter selection.

• Improve Injector reproducibility: Leave the vial cap slightly loose so it does not make a full seal. *This prevents a vacuum forming inside the vial, resulting in injection volumes which may be lower than the selected volume. "Loose caps" can greatly improve accuracy and reproducibility when larger OR multiple volumes are injected from the same vial. Additionally, if the total sample vial volume is very small (i.e. ~ 200 ul), utilize a vial insert of the correct dimensions and type for improved accuracy. When using vial inserts, check that the needle height is correct for the vial insert used.  Do not use the entire sample volume! Never use more than 90% of the vial volume or air may be aspirated resulting in invalid data collection.
  

• Prevent sample carryover problems by regularly inspecting and servicing your HPLC injector (Manual valve and Autoinjector maintenance tips will be found at this LINK). Replace common wear parts such as rotary valve seals and needle seats on a regular basis (Do not "clean" and re-use seals). Carryover troubleshooting Tips will be found at this LINK.
  

•  Calibration Volumes for Quantitation: When creating a new calibration table for a group of standards, use the SAME VOLUME for each standard and vary the concentration ("calibration level") only with each vial. As we have seen, injection volume is a variable which may change peak shape and integration accuracy. If you inject the same volume of liquid for all standards (and samples too), then you remove this variable. Using the SAME injection volume for all standards and samples helps to reduce problems. *Note: Thought it may not be approved, if you thoroughly test varying the injection volume across the range used for the calibration to demonstrate no undesirable changes to peak shape, loss of resolution/separation, and it is reproducible and accurate for the analysis method, then you can vary injection volume. Link to: HPLC Calibration Article.
   

 

Please note that these are general guidelines only and the mode of chromatography (e.g. NP/RP/HILIC/SEC), scale (prep vs. analytical) and/or specific method used must be optimized for best results. Follow these basic guidelines to prevent analysis problems, prolong column and system lifetimes and increase reproducibility and accuracy.