Translator for HPLC HINTS and TIPS for Chromatographers

Saturday, September 5, 2020

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 operates 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 (measure it).
  • 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 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) are within the optimal range of the pump;

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

(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 it, and you will 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 to learn how to use the pump on your specific system. Learn how to run simple verification tests to check the flow rate (with a graduated cylinder). Never rely on the software values, check and verify. Priming and flushing are needed any time air bubbles are present, mobile phase solutions are changed or the system has sat unused overnight. Always flush multi-channel pumps (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).

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. You will need 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 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 or 3x 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 flow rate to speed up the 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 (Flush the Channel).

  • *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 system (i.e. column), just the 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. 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 (piston seals) so if the system struggles to fill with liquid after one minute, discontinue and manually prime.


  • The back-pressure shown on the system readout should be very low during this priming process (e.g. < 15 bars) as the HPLC system should not be plumbed with the column or detector inline, during the priming process. 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.

  • 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.

  • 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, try drawing liquid through this port, while it is running, to gently fill the pump head chamber and remove the air. Alternatively, the outlet fitting above the pump head can sometimes be briefly loosened allowing the system to push the air out more easily (open it slightly with a wrench, then quickly close it). Have a towel ready to soak up any fluid that comes out.
  • The outlet check valve can also become "stuck" open in some cases and drawing liquid out of the pump head's outlet port with a syringe (or pushing it through the pump head) may remove the air bubble and prime the valve, restoring function. 
  • 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). 
  • 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 may result in loss of prime, baseline and 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). 
  • 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 three 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 line, 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 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. Internal damage may result in air or other contaminates leaking into 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.  
  • Additional Troubleshooting Info can be found here:

Diagnosing & Troubleshooting HPLC Pressure Fluctuation Problems (Unstable Baseline)

Saturday, August 8, 2020

Resetting or Clearing the TCP/IP stack to Resolve Connection Problems

  • Warning: These commands should only be used by experienced users who both accept and understand the risks involved. Before using any system commands or utilities, please backup all systems, programs, applications, data and files.
BACKGROUND: A TCP/IP Stack Reset or 'clearing' will flush the computer’s stored Internet Protocol settings back to their default values. Flushing or resetting these values may solve some types of Internet connection issues where screen messages such as; "This Site Can’t be Reached” or “Unable to Connect to the Internet” are observed. 

EXAMPLE 1: To reset the TCP/IP stack in Windows O/S, you can use the Command Prompt. You MUST use and run the Command (CMD) prompt screen with Administrator's Privileges (or it may not work). 

   In Windows 10 O/S, from the CMD prompt screen, type and run the following commands:

  •     netsh winsock reset and press Enter.
  •     netsh int ip reset and press Enter. (*this command rewrites two registry keys)
  •     ipconfig /release and press Enter.
  •     ipconfig /renew and press Enter.
  •     ipconfig /flushdns and press Enter.
  •  Exit out of the CMD screen (exit) and recheck your connection. A re-boot, restart should not be needed.
   In Windows 7 O/S, from the CMD prompt screen, type and run:
  • netsh int ip reset resetlog.txt 
  • Exit the CMD.
  • Restart the computer and check your connection.

EXAMPLE 2: For most MacOS, the TCP/IP stack reset can be run by renewing the DHCP Lease.
  • Click on System Preferences then Network. Click on the Internet connection with status Connected. Next, click the Advanced button. Open up the TCP/IP menu and click on Renew DHCP Lease followed by the OK button.