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 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.

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.

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).

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.

Tips and Advice for Priming your HPLC PUMP (or similar pumps, FPLC or UHPLC Pump)

The single most important component of any HPLC system is the Pump module. We often refer to it as "the heart of the HPLC system". 

• You may have the most sensitive HPLC detector, the best column, a perfect method of analysis, but none of this will matter unless the HPLC pump(s) that provide mobile phase to the system operate perfectly, all of the time. If you have a poor quality (or poorly maintained) system, then you will spend much of your time trying to establish reliable flow through the HPLC system, not running samples. 
• Before using an HPLC system, you should prime all of the lines in your HPLC pump. This is needed to purge any air from the tubing, introduce fresh mobile phase to each line and then to VERIFY that each channel delivers the reported amount of fluid to the column (measure it).
  

• NOTE: This is a LONG, detailed article with lots of information, Hints and Tips. It is available in PDF format for download, here.

The HPLC pump's ability (stability) to provide reliable operation depends on: 

(1) The Chemical, Physical and Miscibility properties of the Liquid(s) being pumped;

(2) The Amount of dissolved gas inside the liquid (must be minimized);

(3) The Temperature of the room (or HPLC) must be stable;

(4) The Position of the mobile phase bottles (relative to the pump, above or below);

(5) The Solvent Pickup Filters used (are clean and appropriate in material & porosity);

(6) The Fittings used are correctly installed & tightened;

(7) The types of Tubing used are chemically, temperature and pressure compatible (esp. the Inside Diameter of the tubing);

(8) The Selected Flow Rate(s) and Back-pressure are within the optimal range of the pump;

(9) All mobile phase solutions are Filtered, Freshly prepared and Degassed;

(10) How often the Pump is properly Inspected, Cleaned & Serviced.

 The HPLC pump is the most important part of your HPLC system. Take care of it. Neglect or abuse it, and you may lose time and money. Almost every problem you experience using an HPLC will be related in some way to the pump. Make sure you understand the flow path of the system in detail, and have the training to setup and use it properly. Take a hands-on training class (not a video or web based tutorial) to learn how to use the pump on your specific HPLC system. Learn how to run simple verification tests to check the flow rate (best done with a graduated cylinder). Never rely on the software values, check and verify everything yourself. Priming and flushing are needed any time air bubbles are present, mobile phase solutions are changed or the system has sat unused (this includes overnight). Always flush multi-channel pumps and valves (i.e. Binary, Ternary, Quaternary...) using a setting of 100% channel composition. Run one channel at a time at 100%, not 25% or 50% to flush channels (a common novice mistake). Flush ALL channels on a regular basis.

OK, so what can you do to make sure your HPLC pump is properly primed with fluid and operating to the best of its ability?

Start, by reading the operator's manual for your pump. Review the procedures for connecting it to the system, become familiar with the flow path and understand the procedures to prime the pump heads. Practice these procedures.

If an inline vacuum degasser is used, become familiar with the specifications, chemical compatibility (some are not compatible with solvents such as strong acids, strong bases, THF, chloroform, fluorinated additives and so on) and internal channel volume of each chamber used. It is useful to know what the degasser chamber volume is to figure out what the total channel priming volume is. This may be different for similar systems. Check, measure, verify, do not assume.

Priming Volume: The total volume contained in each channel's low-pressure line from the mobile phase bottle to the degasser + the degasser chamber channel volume + the total volume in the line from the degasser to the pump head (or multichannel valve) = the total minimum volume you must flush out before using the system. Because flushing just the minimum of volume (1x) of fluid through the channel is unreliable, flush 2x, 3x or more times this total volume, per channel (or as much fluid as it takes), to prime each channel. *If no degasser is present, then just calculate the volume contained in the low pressure tubing from the bottle to the pump head/valve. Set the pump to direct the flow to waste and use a high initial flow rate to speed up the priming process.

Use fresh mobile phase (prepared daily and filtered). Make sure the solvent pickup filters are clean. If possible, have the bottles placed higher than the pump's inlet (once flow has been established, this will allow natural siphoning to push liquid towards the pump head). Prime all of the lines used. The pumps run on liquid, not air so try and fill any of the lines with pure mobile phase before you connect them to the pump and/or degasser (If all of the lines are prefilled with fresh liquid, you can skip this part).  

There are two ways to PRIME EACH line (Flushing the Channels).

• *First, open any Prime/Purge or Waste Valve so the mobile phase is directed to waste, not the injector, column or detector. Our goal is to initially fill the lines with liquid, quickly, and we do not want these fluids to go through the entire HPLC system (i.e. column), just the HPLC pump.

(1) Wet Priming use a syringe fitted with a Luer-to-threaded fitting adapter (usually 1/4-28) to draw liquid through the tubing in the mobile phase bottle and into the pump's degasser and/or pump head's inlet. Be sure to have this type of syringe available (very useful). Never push fluid, only draw fluid through the tubing, just like the pump does. Connect the syringe to the mobile phase bottle lines, degasser ports and/or pump head multichannel valve or pump head inlet, as needed, to draw liquid through until all lines are filled.

(2) Dry Priming using the HPLC pump to draw the mobile phase out of the bottles, through the lines, degasser channels and to the pump head or multichannel valve. Note: "Dry" because the lines (low pressure tubing) are initially dry when we start. Always do this one channel at a time (e.g. A = 100%). This insures no miscibility or mixing problems and is standard procedure. Start with a modest flow rate to get the fluid moving through the lines, then increase the flow rate to speed up the process. The low pressure Teflon tubing is transparent so you can watch this process. Repeat with each channel. Note: Some HPLC pumps will struggle to perform this type of dry priming, as they will be unable to draw the liquid up from the bottle and/or pump the air out of the system. Pre-priming the lines using a syringe (as in #1 above) will help solve this. Running the pump with just air inside the lines may result in increased wear on the system (esp the piston seals) so if the system struggles to fill with liquid after one minute, discontinue and manually wet prime each line.

NOTES: 

• The back-pressure shown on the system readout should be very low during this initial  priming process (e.g. < 15 bars) as the HPLC system should not be plumbed with the column or detector inline, during the priming process (it should be by-passing those parts). Only the viscosity of the solution, the selected flow rate and the internal diameter of the tubing going into and out of the pump will contribute to the observed back-pressure, and this should be very low value.

• Once you have verified that liquid is exiting through the pump head waste port, you can increase the flow rate to speed up the priming process, but pay attention to the back-pressure. It should increase as the flow rate increases and drop as the flow rate drops. Continue to prime each channel in this way, one-at-a-time, until all channels are primed and flushed with liquid. You can gradually slow the flow rate down as you stop, to transition from one channel to another.
  

• If liquid has been drawn to the pump head, but the pump head still is not pumping liquid through it, it may be experiencing cavitation (air locked). If there is an outlet port on top of the pump head, the outlet fitting above the pump head can sometimes be briefly loosened with a wrench, allowing the system to push the air out (open it slightly with a wrench, then quickly close it after liquid comes out). Have a towel ready to soak up any fluid that comes out. Keep the area clean and dry. Alternatively, try drawing liquid through this port, while it is running, to gently fill the pump head chamber and remove the air.

• In some case, the inlet or especially the outlet check valve(s) can also become "stuck" open. When buffers are left in the system (they should be flushed out with water), crystals and particulate matter may deposit on the valve resulting in poor sealing, leaks or air being drawn through. Drawing liquid out of the pump head's outlet port with a syringe (or gently pushing it through the pump head) may remove the air bubble, debris and prime the valve, restoring function. Note: If needed, shut down the pump and clean/replace any contaminated or worn check valves before proceeding.
  

• In more extreme cases, you can change the mobile phase going into the pump head to a more viscous intermediate solvent to get things moving (an alcohol such as IPA might work well. If buffers have been used, then always first flush with pure water). 

• Degas all eluents / mobile phase solutions used. All of them. Degassing will help reduce the formation of bubbles inside the pump head. Failure to properly degas the solutions used may result in loss of prime, baseline and/or pressure instability. Make sure your degasser is operating properly (electronic vacuum degassers only last ~ 5 years at most. Be sure to have them professionally serviced). Sonicating fluids at the bench or using vacuum filtration to initially remove gas from the solution will only degas the solution for a short time (minutes). Gas will slowly diffuse back into the solution resulting in baseline noise, drift and pump problems (for HPLC, only use inline degassing or Helium sparging).
  

• Verify the flow rate. It may be unwise to rely on the indicated flow rate shown on the instrument screen or display. It is wise to measure the flow rate of each channel, separately, using a graduated cylinder and a timer. This is the most reliable way to determine what the actual flow rate is through the system (and is also the method we use during performance verification or qualification testing too). To check the flow, make sure the system has been primed and flushed. Install a flow restriction capillary in place of the column (to provide the required back pressure). Set the flow rate to a value which is appropriate for the pump and measure/record the volume delivered vs. time. Example: Using a flow rate of 1.000 mL/min obtain a 10 mL volume, glass laboratory grade graduated cylinder. At time zero, direct the flow from the restrictor's outlet into the graduated cylinder. Measure the volume of fluid collected in 8 minutes. *It should be 8.00 mLs.
  

If you continue to have priming problems and/or air bubbles disrupting the flow there are four more things you can check. 

1. Make sure the solvent pickup filters/frits are clean and unobstructed (these are maintenance items). If the filters are obstructed, then a vacuum may form on the line resulting in pump cavitation and loss of prime. One quick way to check if this might be a problem is to remove the suspect solvent pickup filter from the tubing, then try again. If flow is restored w/o the filter in place, then the filter may have been clogged. Install a new solvent filter as soon as possible. *Never run the HPLC without solvent filters installed. Those filters perform a very important job and protect the flow path of the system.  
2. Service the Pump Heads. Regular cleaning, inspection and replacement of worn parts must be done to maintain the function of the pump. Worn parts will result in failures, instability, lost time, plus invalid data. The pump has many mechanical parts which wear out and require replacement. Most pumps should be inspected/serviced every 6 months. Keep the pumps clean and fully serviced (replace: piston seals, pistons, frits, check valves as needed). Depending on the brand, model and applications, the types of parts needed and the frequency of repairs varies widely. *This is discussed in another article. 
3. If your HPLC system has an inline vacuum degasser (either a standalone or integrated module), it may be damaged, contaminated or broken. The typical service life of an electronic inline vacuum degasser is only five years (some models have even shorter lifespans). Degasser's with internal damage may result in contamination of the mobile phase. A failing or damaged HPLC vacuum degasser may directly contribute to analysis problems (ghost peaks, pressure instability, poor baseline stability...). Have your degasser professionally diagnostically tested and serviced often.  
4. Clean and/or replace any worn or damaged inlet or outlet pump head valves. Not flushing buffers out of the HPLC system on a regular basis or remain in contact with the solution for long periods of time can damage the valves. In some cases, cleaning is all that is needed, but in others, replacement is required to restore function. Be sure to have the system professionally serviced on a regular basis.
   

• Additional Troubleshooting Info can be found here:

Diagnosing & Troubleshooting HPLC Pressure Fluctuation Problems (Unstable Baseline)

HPLC Column Cross-Sectional Area and Scaling

Here is a simple formula to use when scaling up or down Internal Column Diameter to maintain retention values (under constant linear velocity). Flow rate must be adjusted to account for any changes made to the column's cross-sectional area. We usually refer to these types of changes as the "Scaling Factor". To determine the scaling factor, we need to know the internal column diameters of the two columns we are scaling from (actually, we need to know the radius, but once we have the diameter, we simply divide the diameter by 2 to obtain the radius). *In this discussion, changes in cross-sectional area are the only parameters we are concerned with as column length does not affect scaling.

• Scaling Factor = (S);
• Column #1 Radius =  (R1);
• Column #2 Radius =  (R2).

     S = R2² / R1²

Example #1: 250 x 4.60 mm column scaled down to a 250 x 2.10 mm column. 

          Answer = 0.208. 

• If the original flow rate was 1.000 mL/min, the the scaled down flow rate would be 0.208 of the original or 0.208 mL/min for the 2.10 mm ID column. *For practical use and application, we often use either 200 ul/min or 210 ul/min to simplify the value.

Example #2: 250 x 4.60 mm column scaled up to a 250 x 10.00 mm ID semi-prep column.

          Answer  = 4.726. 

• If the original flow rate used was 1.000 mL/min with the 4.60 mm ID column, then we would increase the flow rate to 4.726 mL/min on the 10.00 mm ID column to maintain the same relative velocity (and relative retention). *For practical use and application, we often use 5 mL/min to simplify (round off) the value. 

Notes:

1. Flow rate optimization should always be carried out by running a standard at different flow rates and plotting the plate height (N) vs the flow rate. Test flow rates that are slightly below the predicted linear velocity and up to 2 times higher than that rate to find and optimize the flow rate for your sample (it must be determined through experimentation for your specific method). 
    
2. HPLC Columns packed with sub 2 micron supports may have optimum flow rates 2 to 5 times more than the predicted std linear flow rate so actual testing is critical to determining the most efficient flow rate. I recommend optimizing the flow rate used with analysis methods which use any particles which are 2.5 microns or smaller in diameter.