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.
   

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.

Agilent Quaternary Pump (e.g. G1311A ) "Secret" Operator Tip to FLUSH the HPLC Pump in 1/2 the time!

One of the most popular "tips" taught in our Agilent 1100 and 1200-series HPLC training classes shows users how to speed up the daily priming and flushing process of the Quaternary Pump. Many people use these pumps without taking advantage of the Quaternary pump's higher flow capability. If you are not currently using the higher 10mL/min flow rate capability offered by this pump (vs. the Binary pump's 5 mL/min), then you are missing out on a free time saving feature. Please read on to learn how to use this feature.

Based on the HP 1050 pump and introduced in 1995 as the "1100-series" version, the G1311  "Quat" pumps are one of the most popular research grade HPLC pumps found in laboratories today. They are extremely reliable, rugged, easy to operate and service. The Quat pump is driven by an easily accessible, single pump head with an in-series, servo controlled dual plunger and Multi-channel Gradient Valve ('MCGV') for 4-channel solvent proportioning with an active inlet valve (known as the 'AIV', first used in the HP 1050 pump and the reason for this pump's high reliability. No more "sticking" inlet valve issues!). Unlike the Agilent Binary pump (G1312), which uses two separate dual plunger pumps (2-channel) at up to 5.0 mL/min (maximum), the Quat pump offers an extended flow range, up to 10.0 mL/min (maximum). However, most users are not aware of this or do not know how to utilize this higher flow rate feature because the Quat pump defaults to a maximum flow rate of 5 mL/min at initialization. The ability to program the pump to operate at flow rates greater than 5 mL/min requires a "trick" to activate it (which apparently is a secret as we rarely encounter customers who are aware of how to use it). 

Let me share with you why you would want to use this feature, why the feature is hidden to most and of course HOW TO ACTIVATE IT on the Quat pump.

• Q: Why would you want to run the pump at 5 to 10 mL/min? Semi-prep columns can be run within this flow rate range, but a more common reason to operate at 10 mL/min is for daily system start-up. Anytime you replace or change the mobile phase bottle/solution OR when you startup the HPLC system (each day) one of the very first things you need to do is prime or flush each of the mobile phase channels, one-at-a-time through the system to waste. Air bleeds into the system when it is not used and this procedure primes the lines and pump head with fresh mobile phase preparing it for use. The system's flow path is directed to waste (via the open, prime-purge valve) during this step so back-pressure is not a concern. The higher the flow rate you can use for this flushing step, the sooner you can complete it. If you run the pump at 10 mL/min vs 5 mL/min, then flushing can be completed in half the time. This is especially useful if you have a model G1322A degasser module installed as the internal volume of each degassing channel in the G1322A is 10-12 mLs, requiring extended flushing times (4x channels = 30+ mLs flush per channel) before moving on to the next channel.

• Q: Why does the Quat pump initialize with a reduced, 5 mL/min maximum flow rate? The Quat pump was designed to meet two different operating pressure ranges. From 0 to 5 mL/min the permitted operating pressure range is 0 - 40 MPa (0 - 400 bar). Above 5 mL/min, the operating pressure range is reduced, 0 - 20 MPa (0 - 200 bar). As most analytical chromatography is performed at flow rates below 5 mL/min, the system initializes using the more practical, 0 - 400 bar range, limiting flow rates to 5 mL/min maximum. The default maximum pressure field is set to 400 bars. You should always change the maximum pressure value from 400 bars to a more realistic maximum pressure (lower value) for your method. Use a maximum value that is appropriate for your own method. *The only time you will want to set it to the maximum value is when conducting a Pump Pressure/Leak test (it must be set to max pressure for testing).
  

• Q: When I try and enter a pump flow rate larger than 5 mL/min, the system does not accept it. How do I program the pump to increase the flow rate past 5 mL to 10 mL/min? In order for the system to accept a flow rate of greater than 5 mL/min, you must FIRST set the maximum pressure limit to a value that is 200 bars or less (within the allowed "0 - 20 MPa (0 - 200 bar)" range). Once the maximum pressure limit has been reduced in the method, the system will then allow you to enter a higher flow rate such as 9.999 mL/min (10 mL/min). As long as the maximum pressure alarm is set within this window (200 or less), the pump will allow flow rates above 5 mL/min to be used. Now you can program the pump to flush lines or prime the system at twice the speed of the Binary pump equipped systems (10 mL/min).

Please share this "trick" with other users of the G1311A, G1311B, G1311C versions of this pump so they can maximize their time and productivity. Let us know if you find this tip useful.

Adjusting the HPLC Gradient Time For Changes in Column Diameter and/or Length (same particle size)

Changes to the column diameter (to scale the method up or down) can be calculated. For an established HPLC method using the same support type (same exact material and particle size) where the column dimensions and flow rate are known. Note: If only the diameter changes and the lengths remain the same (proper linear flow rates used in both cases), then the resulting gradient times will also be similar. If the column lengths change, then the gradient time will change.

Changes to the Gradient Time (Tg2) used for a second column which has a different diameter, "Dc2" and/or length, "Lc2" can be calculated if you know: 

• Tg1 [Time, of initial Gradient on Column #1];
• Tg2 [Time of second Gradient on Column #2];
  
• Fc1 [Flow Rate of Column 1] ;
• Fc2 [Flow Rate of Column 2];
• Dc1 [Diameter of Column 1]
• Dc2 [Diameter of Column 2];
• Lc1 [Length of Column 1];
• Lc2 [Length of Column 2].

        Tg2 = Tg1 x (Fc1 / Fc2) x (Dc2² / Dc1²) x (Lc2 / Lc1)

 

Example: Initial Method utilizes a 4.6 x 150 mm, 5u column run at 1.00 mL/min with a 10 minute gradient program and we wish to transfer this gradient method over to a column with a 2.1 mm diameter (ID) x 100 mm column run at 200 ul/min.

   Tg2 = 10 x (1 / 0.2) x (2.1² / 4.6²) x (100 /150)

   Tg2 = 10 x (5) x (4.41/21.16) x (0.67) 

   Tg2 =  50 x 0.208 x 0.67

   Tg2 =  6.97 minutes.

The gradient time used on the 2.1 x 100 mm column run at 0.200 mL/min would be ~ 7 minutes (vs 10 minutes on the 4.6 x 150 mm column at 1 mL/min).

 

NOTE: A note about optimized flow rates. If the Column PARTICLE SIZE changes, esp from greater than 3.5 u to less than 3.5 u, then the optimized flow rate may also change too. Please refer to my article; 

• "Speed Up HPLC Analysis Time Using Higher than "Normal" Flow Rates with SMALLER Particles" for more information.

GC-MS Contamination Identification and Sources

Over the past few months I have received a number of questions regarding the identification and possible sources of contamination observed in GC-MS systems (GC/MSD, EI). Contamination can be sourced to the instrument itself or to materials used in the preparation of samples. We shall review a few of the most common types of contamination observed and how you can use the MS detector data to identify them in this article.

Air Leaks / Contamination: The GC-MS detector operates under vacuum conditions so air must be kept to a minimum detectable level. 

• Common Sources: Improper fitting connections; improper fitting materials/age; damaged or worn vacuum seals. 
• Identification: High background noise levels (poor S/N of stds); Peaks associated with air (i.e. m/z 18, 28, 32, 44).

Acetone Cleaning Fluid Contamination: Often used to clean the metal parts of the source, acetone may appear in the signal. Look for peaks at m/z 43 & 58.

Dimethylpolysiloxane Contamination/Bleed: Silicone from the various seals and septa may appear and depending on the chemical source, are often detected at m/z 147, 207,221, 281,295,355 & 429. *Always use high quality seals & inject blanks often to check for contamination.

Plasticizer Contamination: Many plastics are used in the seals found in the GC-MS instrument. These plastics may eventually bleed into the system and be detected (due to normal wear, heat stress or even poor sample preparation). It is critical to identify which ones result from worn out seals vs. use of plastics as part of improper sample preparation procedures (e.g. improper handling techniques, glove material, plastic sample tubes, washing glassware with soaps etc). One of the most common peaks observed will be m/z 149 (Phthalate).

Diffusion Pump Oil Fluid Contamination: Turning off the carrier gas flow may allow for some of the diffusion pump oil to back-stream. *Maintain carrier gas flow when this system is ON. If diffusion pump oil has entered the source, you should observe a strong signal peak (e.g. m/z 262, 446 for the oil).

Foreline Pump Oil Fluid Contamination: Look up the specific chemical composition of the oil used to obtain applicable m/z values to check for (e.g. hydrocarbons, m/z 69 ...).

*Avoid GC-MS contamination and trouble by first receiving the proper training to operate, use and maintain the GC-MS system. This includes making sure the entire system is fully maintained, seals changed, performance monitored regularly, insuring all of the vacuum pump oils are changed on a regular schedule and always use high quality replacement parts. Maintenance is something that most users can perform themselves, especially if they have completed a formal hands-on training class using their own GC-MS system (Most manufacturers offer basic on-site Maintenance classes).

Troubleshooting HPLC Gradient Valve / Proportioning Valve / MCGV GPV Leaks. How to Identify Them.

HPLC pumps which utilize low-pressure mixing VALVES are known by names such as: "Ternary" (3-solvents) or "Quaternary" (4-solvents) pumps.These types of HPLC pump configurations use a single, high-pressure pump head coupled to a multi-port / proportioning valve and represent some of the most popular and versatile pump configurations offered. Featuring random access to multiple solvent bottles (more than two is always better), lower operating costs and less maintenance work provides you with one of the best platforms to develop new methods on. I highly recommend them for most, but not all, HPLC applications (vs. Dual pump, high-pressure "Binary Pumps").

• If your HPLC system utilizes a single, high-pressure pump head coupled to a multi-port valve, then please remember that in addition to pump head maintenance, regular maintenance of the multi-port / proportioning valve is also required.

A few weeks ago I was hired by well known Pharma company to solve a gradient method problem that I was told has stumped their best scientists for almost one year. The client presented me with their validated UHPLC method which suddenly developed a shift in retention time of all peaks. The shift was significant, about 10% of the previous values over a 20 minute run, and had been observed on two different, but similarly configured HPLC systems in their lab. Changing the column to a new one showed no change on either HPLC system. They were out of ideas.

• Before I reveal the cause of the trouble, let us briefly think about what types of changes can result in a small, repeatable shifts of peak retention times. Four common ones that come to mind are: 

(1) Flow Rate changes;

(2) Column Temperature changes;

(3) Column Fouling;

(4) Mobile phase composition changes. 

Start the troubleshooting by ruling out the easy causes first (#1, 2 and 3 above).  

• (1) Flow Rate: When the actual flow rate is in question, start by measuring it manually Never trust the instrument's display screen value or the software's 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, then using water, measure the time it takes to fill a 10mL graduated cylinder to the 5 mL line. It must take exactly 5.00 minutes (= 1.00 mL/min). Run this flow test on each pump channel.
  
• (2) Temperature: The HPLC method should be run under controlled column temperature conditions. Verify this. Retention times are a function of temperature (i.e. cooler temps usually result in longer retention times, warmer = shorter). The temperature should be stable (~ 1 or 2 degrees C).
  
• (3) Column Fouling: To prevent fouling, wash the HPLC column with a solution that is STRONGER than the mobile phase after each analysis. Use fresh, clean solutions. Verify that the samples are dissolved in the mobile phase (100% dissolved) and filtered before injection. Verify that the injection volume is less than ~3% of the column volume and the concentration of the sample is not too high (avoid saturating or overloading the column). Solubility is very important for both the sample and any additives used in the mobile phase (to prevent precipitation). Anything that "fouls" the column support will directly effect the retention times and often the peak shape too. Be aware of these causes and take action to avoid them.  *Replacing a suspect column with a new one is often an inexpensive way of troubleshooting a "peak" problem. Always have a NEW spare column on hand for testing. *Columns are consumable items.
  

• (4) Mobile Phase: Changes to the actual amounts of additives, pH or final composition of the mobile phase may impact peak retention times (sometimes, the peak shape too). After all, the final composition used was developed for the purpose of establishing a reliable and reproducible method of analysis. It must be controlled. We must take steps to insure the mobile phase preparation and delivery are accurate. Always prepare fresh solutions each day (esp. all aqueous solutions!). pH values may change after a few days (e.g. even in MeOH / acidic solutions), bacteria/mold/algae grow quickly in many solutions, even in the refrigerator, so only prepare what you need for the day. Evaporation of more volatile solvents (in pre-mixed solutions) can change their actual concentration (always protect them from heat and evaporation).
  

*There is another way that the mobile phase composition can change which often goes unseen. It can change during delivery to the column. The HPLC's low pressure proportioning valve that allows us to easily select and use different solvents can develop small internal leaks, resulting in valve cross-flow leakage. This cross-flow leakage allows liquid (or air, if the line is not connected) to be drawn out of one channel and into another, changing the actual mobile phase composition. This happens because the valve seals, esp if they have been left unused for a long time, can change shape (e.g. shrink) and begin to leak over time. Often the amount of leakage is very small (ul/min), but depending on the method, a small change may result in a significant change to the chromatography.

I reviewed the client's method parameters and concluded that the method met good chromatography fundamentals. Checking the flow rate (using a graduated cylinder) confirmed the flow rate was accurately shown. A review of their mobile phase preparation procedures and methods also appeared OK. Degassing of mobile phase and column temperature were also satisfactory. 

As I looked more closely at the two running HPLC instruments they used, I began to quickly zero in on the most likely problem. 

• A long stream of air bubbles were observed exiting the HPLC pump's gradient valve leading into the high pressure pump head, but no air bubbles were seen exiting the degasser's outlet line (IOW: The vacuum degasser may or may not be the cause, though it is critical to insure the degasser is clean and fully serviced before use. Have the degasser professionally serviced first before proceeding with troubleshooting. Using a damaged degasser will make it difficult to use the pump or run any valid tests as degassed solution is needed). This was observed on several of their HPLC systems, including the two used for this method. The fittings connecting the lines from the degasser module to the valve were correctly connected (as a loose connection would cause air to leak in and must be quickly ruled out). 

The cause was from one or more of the unused gradient valve positions leaking air into the flow path, changing the mobile phase composition. Of four possible mobile phase lines available (A,B,C,D), the client only had two lines connected to mobile phase bottles (A,B) with the remaining two lines left open to the air. The internal valve seals in the unused 'C' and 'D' valve positions had deformed, shrinking in size, sticking,leaking, allowing air to flow into the mobile phase on one of the channels. This resulted in a change of the organic composition % used in the method (due to a cross-flow leak), changing the peak retention times (as the actual mobile phase composition used in their gradient was different). I directed the HPLC pump's outlet line to waste, placed all of the solvent pickup bottle lines (A,B,C,D) in a beaker filled with IPA and allowed the pump to run pure IPA at 1 mL/min across each channel, one-at-a-time (100%), for ~ 20 minutes to re-hydrate the internal gradient valve seals. This was repeated with each valve position, then all of the lines were placed in fresh mobile phase solution, primed and flushed. The system was restarted and the method now ran showing the expected peak retention times. Instructions were provided which included regularly using all of the channels and valve positions plus flushing weekly to maintain valve operation. Use ALL of the lines and flush the valve(s) through all positions, one-at-a-time, on a regular basis. If prolonged flushing with pure IPA does not fix the leak, then it is time to replace the valve. All valves eventually wear out and must be included in maintenance inspections and checks. This is especially true when you purchase your HPLC system at an auction or from an 'equipment' reseller. Never assume that the 10+ year old HPLC valve is OK. Test it first (e.g. Acetone tracer test).

 

Acetone Tracer Test: If you suspect that a cross-flow leak exists on a gradient valve, then one method I use to check for leakage is to mix up a "Tracer" solution of pure organic (often ACN) that has 1% Acetone mixed in (for RP methods). Remove the column and replace with a restriction capillary. Place the tracer solution on the valve position you suspect may be leaking at an appropriate flow rate and set it for 0%. Run one of the other channels with 100% (pure ACN in this example) and monitor the UV (265nm) for the presence of acetone. If the acetone leaks into the channel you are using, it will be easy to observe on the UV trace. So called "bubble" tests (introducing and monitoring the position of a gas bubble into the low pressure solvent line) are not reliable leak detection methods for small leaks. Use a tracer such as acetone to find the leaking channel(s). You can read more about these types of Valve Leakage tests in this article (Click Here).

Repair Corrupted Windows 7, 8 & 10 System Files Using the System DISM Tool

Time to share another useful Microsoft Windows command-line utility tool. If you have experienced the "Blue Screen of Death", a crash, service pack installation failure, windows update failures or observed general Windows system file corruption error messages, then this utility tool might be useful to you.

Many common Windows Update errors which result from new system file corruption can be corrected using the Windows DISM.exe tool. Failed application or update installs often result in corruption of some of the system files. These utilities are designed to detect corrupted file and repair them. It targets the currently running operating system for repair. The DISM tool must be run from the Windows Command prompt (cmd), with an account that has administrator privileges using " Run as administrator ".

DISM = Windows "Deployment Image Servicing and Management" tool.

• Before using any software utility program, make sure you first have permission and authorization to do so. Most Importantly of all: Back up your system programs and any data files before using any utilities such as this one. Create a Restore Point to protect the basic settings too. Do this now. Take precautions before using any utility programs and do so at your own risk. You are responsible for your data, programs and computer.
  

• Please make sure you have reviewed my earlier article on how to run the System File Checker (SFC) tool first. The SFC tool scans Windows operating system files for corruption AND restores any found corrupted files, all automatically! The SFC often quickly corrects many system errors and I always run that utility first. DISM is more thorough, but takes more time to run.

• Before using the DISM (or SFC) utilities, set a new restore point using the very useful "Restore Point" feature found in Windows (discussed in an earlier post). Make sure you have enough time available for the computer to run this utility (overnight is best). Once started, it will show a progress bar. Do not interrupt the process.
  

To run the DISM.exe utility, close down all applications for now. Make sure your account has Administrator privileges, then open up the Command Prompt using the " Run as administrator " option (you must do this so the system32 path is selected). 

At the command line prompt, Type the command line below (make sure to include the spaces, exactly as shown): 

 DISM /Online /Cleanup-image /Restorehealth  

 Optionally, for some O/S enter:  DISM.exe /Online /Cleanup-image /Restorehealth 

The screen should show " Deployment Image Servicing and Management Tool " with the version #. Image Version plus some [ ] showing the progress. When it is finished, it should report that "The operation completed successfully". Type 'EXIT' to close the Command Prompt screen, then Reboot your computer.

 

 

References:

1.  Microsoft Windows Support Page:

Fix Windows Update errors by using the DISM or System Update Readiness tool 

 

2.  If you encounter "error 87", then please confirm you have: typed the command line as shown; are running the cmd "as administrator"; have applied all new or pending Windows Updates and have run the SFC tool first. If you continue to see the error, then refer to Microsoft's knowledge base for additional tips.

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.

Capillary Electrophoresis (CE) Troubleshooting Tips:

What follows is a short list of problems, "observations" followed by a list of areas that should be investigated, as appropriate in parenthesis (), to troubleshoot common problems seen when using the analytical technique of capillary electrophoresis, CE, CZE.

 Observation (Investigate for cause):

            Excessive Baseline Drifting up or down

·         Temperature is not stable (stabilize room and/or capillary temperature).

·         Fouling of capillary (replace or clean and wash capillary with fresh, filtered solution).

·         Current levels unstable (loose connections, partial obstruction in capillary or running out of buffer solutions).

·         Capillary may have poorly cut ends resulting in poor connections or flow (replace capillary).

Excessive Signal Noise

·         Detector has air in flow cell (purge capillary and wash flow path).

·         Current level may be too high (reduce current).

·         Detection parameters, wavelength and bandwidth, may be inappropriate for buffer solution (select appropriate detection settings which are appropriate for the buffer used and selective for the analyte).

Loss of Signal

·         Voltage/Current has turned off (turn ON or investigate if system is in “alarm” state due to an error).

·         Detector parameters not selected.

·         Capillary has not been fully equilibrated (equilibrate capillary and auto-zero the scale).

·         Baseline offset may be off-scale (after equilibration, adjust scale or auto-zero).

·         Detector lamp(s) off, not ignited or due for replacement (verify lamp operation).

Signal Peak Shape Issues

·         Truncated, clipped or ‘square’ peaks (sample overload, reduce concentration 10x, shorten load time and re-evaluate).

·         Tailing peaks often result from very high current or when the concentration of buffer is too high (lower the current and/or reduce the buffer concentration, then re-evaluate).

·         Sampling rate may be too low (measure the peak width in units of time (i.e. seconds), then configure the detector to insure that the sampling rate allows for at least 20 points to be collected per average peak width (30 points is a better target # to use).

·         No peaks observed (Many possible causes, including: Partially or fully obstructed capillary, broken capillary, out of buffer, no injection, detector settings inappropriate for analysis, current too low, pressure too low. Look for a small peak from the injection along the start of the baseline to confirm that an analysis was started, then troubleshoot the method and settings).

            General Stability and Noise Issues 

·   When the CE system has not been used in a few days, salts from the buffer solution(s) may deposit on and clog the capillary line, flow cell and/or sensors. To avoid these problems, be sure to thoroughly clean, flush and wash down the flow path before use. Take the time to prepare fresh filtered solutions (each day) and allow time for the system to equilibrate. Taking these basic steps will avoid many hours/days of frustration.