- 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:
- *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.
- 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.
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.
- 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.
- 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.
- 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: