More Technical Service “Red Flags” – LC

red flagThis post is the second of its kind pertaining to LC analysis, all of which are an extension of a series pertaining to “Red Flags” for GC analysis. These are situations and symptoms that tell us in the Tech Service group that something is just not right. As we discussed in the first post, Technical Service “Red Flags” –LC,  here are more examples of some “Red Flags” that we sometimes see for HPLC:


Loss in retention:

Customer reports a dramatic loss in retention for all analytes, which elute at or near the void volume.

A secondary complaint with this scenario may also be asymmetrical peak shape, although it may not be noticeable because peaks are often hard to distinguish at all. This is usually caused by a phenomenon known as phase collapse. Here is an example taken from one of our technical articles:


phase collapse


Phase collapse, chain collapse, or dewetting, as it is sometimes called, is the result of exposing a column to a mobile phase with very high aqueous content. A conventional C18 column is not intended to be used with higher than 95% water.  What happens is that the mobile phase is so polar that it is repelled by the nonpolar C18 chains and cannot penetrate between the chains. The aqueous solvent will initially fill the pores of the particle, but as soon as the pressure is lowered or the solvent pump is turned off, the water is forced out. The chains then collapse and lay down flat against the surface at this stage. Usually the problem with retention is noticed after the pumps are turned back on and when the next sample is injected thereafter.

The good news is that usually the phase can be rewetted by flushing the column. First make sure any buffer is removed by flushing with mobile phase that contains no buffer salts or modifiers for several column volumes. Then follow that with 100% organic solvent (acetonitrile is preferred). You should able to switch back to your regular mobile phase after that, just make sure that you use no more than 95% water. If your application requires a higher percentage of water in the mobile phase, make sure that you use one of our Aqueous C18 column phases, which include the Ultra Aqueous C18, the Pinnacle DB Aqueous C18 and the Allure Organic Acids columns or a polar embedded column such as our Ultra IBD or Pinnacle DB IBD column.


Visible white residue:

Customer reports visible white residue or particulates coming from the outlet of the column

If this residue does not resemble anything from the samples you have been injecting, then this could be packing material coming out of the column. It is important to STOP the pump flow immediately and disconnect the column outlet so that these particulates do not clog anything downstream of the column in the flowpath. Eventually, this will result in some changes in pressure. Pressure will increase if a clog develops in the flowpath after the material exits the column. If that does not happen (or before it happens), you may actually see a decrease in pressure, deteriorating peak shape and/or a loss in retention of analytes.

The first thing to check when you see this residue is the pH of the mobile phase. If using a pH meter, please confirm that the device is calibrated properly. At some point above a pH of 8.0, the silica may begin to dissolve and when the particles become small enough, they will start coming through the frit at the end of the column. If pH is not the issue, check to see if you have the column installed with the proper direction of flow. Some of our columns are intended to be used with the flow in one direction and cannot be reversed, even for cleaning purposes. This applies to our Pinnacle DB UHPLC (1.9 µm) and our Raptor™ columns. Please look for the arrows on the column that indicate direction of flow to confirm the orientation. Leakage of packing material can also occur if end fittings in the column have been tampered with.  For this reason, we do not recommend disassembly of our columns by the customer. If the column is not assembled properly, or the frit has become dislodged, leakage will likely occur. If packing material has escaped from the column, it has been damaged beyond repair and must be replaced.

If, by chance, you find material of this nature is coming instead from the guard cartridge, please stop the pump flow and disconnect the analytical column to minimize damage. Then carefully flush out all of the tubing and fittings before installing a new cartridge.


No peaks at all:

Customer reports no detection of any peaks.

Occasionally we may hear from an analyst with this issue in their lab. For troubleshooting suggestions, please review the earlier blog post Troubleshooting HPLC-Loss in Response for All Analytes. Although very similar to the scenario from the earlier blog post, the following possibilities are more likely when you are seeing no change in baseline at all.:

  • Detector is turned off or lamp is not on.
  • Incorrect setting of wavelength on detector or attenuation setting is way off.
  • A solvent pump is not working properly; flow rate is way too low or no liquid flowing at all.
  • No sample is being pulled up for injection or the sample injection (rotary) valve is no longer working.
  • Tubing is plumbed incorrectly or there is a very large leak somewhere in the system.
  • Sample/standard is not prepared correctly.

If you have ruled out the above possibilities and you still feel the column is at fault, try using the same column in a different HPLC system or try using a different column with the same system. Even when column condition is poor, usually you will see at least a solvent peak or disturbance (“blip”) in the baseline around the time the sample is injected. Switching the column or system should shed some light on the situation. Also, please be sure to check out the video in our library “LC Troubleshooting: Baseline Problems”.

Please note that it is important to address one thing at a time, so that you can learn the root cause of the problem. If you still need assistance, feel free to contact us in Technical Services.

I hope this has been helpful and thank you for reading.



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