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Technical Service "Red Flags" - LC

  • Nancy Schwartz
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This post is an extension of a series of posts pertaining to “Red Flags” my colleague has written, pertaining to GC analysis. These are situations and symptoms that tell us in the Tech Service group that something is just not right. So what are examples of some “Red Flags” that we commonly see for HPLC?  Below are some of the most common ones that we encounter:


HPLC column high pressure:

Customer reports rapidly increasing pressure with or without a guard column attached. With a guard, the complaint may be that it needs to be replaced frequently. 

Our first recommendation will be to isolate and confirm the source of pressure, as described in the blog post Building up pressure on HPLC.  First and foremost, it is critical to distinguish between the pressure of the LC system itself versus the columns and guard holders attached.

If using a guard, we always ask if the pressure returns to normal when you remove guard cartridge and/or cap frit inside the holder. If it does, then that confirms that pressure is coming from components of the sample that are being injected. In rare cases, harmful components may also come from mobile phase.

We typically ask what sample prep procedures are used. Samples must be filtered to avoid such issues and if mobile phases contain buffer salts, they must be filtered also. If samples are not filtered with either a syringe filter or Thomson Single Step filter vial, we cannot guarantee the lifetime of a column, guard cartridge, or inlet filter.

If you have purchased a column that can be flushed in a backward direction (3 µm and 5 µm particle size columns), then you may be able to help repair the damage that has occurred to the column. Please follow the instructions located here on our website:

LC Cleaning Recommendations


HPLC column degrading peak shape, often with pressure increase:

Customer reports rapidly deteriorating peak shape, usually with increased tailing and broadening peaks, resulting in loss of resolution. Increased pressure is sometimes, but not always reported as well. Shifting retention times are also sometimes observed.

Similar to the previous scenario, if using a guard, we always ask if peak shape returns to normal when the guard cartridge is replaced or when the cartridge and holder are removed. If it does, then that confirms that materials from the sample are likely affecting the packing material. This can be a physical change due to particulates or a chemical change due to fully dissolved sample components (such as in urine matrix). If the initial issue was accompanied by a pressure increase, it is likely a combination of both factors. In either case, please DO continue to use a guard cartridge as it is protecting the analytical column which is much more expensive to replace. Also, please do try replacing the guard cartridge more often. Once a guard cartridge is saturated with chemically active species, it will begin to spill over onto the analytical column. Thus, replacing the cartridge BEFORE you notice a decline in peak shape will protect the analytical column much better.

Again, we typically ask what the sample prep procedures are. Filtration of samples will prevent damage due to particulates, but does not prevent damage from chemically active components in the sample. These must either be captured by a guard cartridge and/or removed by a cleanup procedure. Techniques such as liquid-liquid extraction, Quechers or SPE are all useful tools for this. Selection of the best technique depends on your specific application. The most efficient procedure will be the one with that accomplishes the objective with the most ease and fewer steps.

Please note that although you may be able to determine that sample components are adversely affecting your column, often the damage due to chemical changes is irreversible. Sometimes flushing the column as described above will help and is worth a try. The reason it helps is because of the introduction of solvents with varying polarity, which may remove some bound substances that would not normally be removed with the gradient you usually use.


HPLC column high pressure with Raptor™ 2.7 µm:

Customer reports excessive pressure starting from when the column was first installed on an LC system.

Some HPLC systems have a 400 Bar pressure limit and are not appropriate for use with 2.7 µm particle size Raptor™ columns. This includes some models that are very common in the field, most of which are more than a year or two old. This is discussed in the post: Should I use a 2.7 or 5 µm Raptor™™ column?

If you do not have an LC system with a pressure limit of 600 Bar or higher, we suggest you try using a 5 µm particle size Raptor™ column.


I hope you found this information useful. Thank you for reading!

Be sure to check out the next post in this series here:

More Technical Service "Red Flags" -LC




Tue, Feb 06, 2018

Hello Garry, I'm glad the application is working well so far. There is not a problem with running at 350 Bar; it is just a matter of your comfort level operating somewhat close to the limit of your instrument. Usually as a column is used the pressure does gradually increase, so eventually you might get an overpressure error. That being said, if you are carefully filtering all of your samples and the matrix is fairly clean, you may be able to operate for a relatively long time in this manner. Using a guard cartridge and replacing it frequently usually helps also. An easy way to lower your pressure just a little bit is to lower the flow rate. I would suggest that if you want to use a method that will work for a long period of time for you.

Tue, Feb 06, 2018

We are using some 2.7 micron columns with an Agilent system, that has Pmax=400. For these experiments, there are major advantages to running at 350 Bar. The results look very good, so far, and we want to continue with this. Will this pressure cause increased wear on the pump, esp piston seals, motor, etc? We have considered buying a UHPLC system (Pmax =800 or even 1200), but the cost is quite significant for that technology. With those pressure limits, we would not be near Pmax, which might be a good idea. Is there really any reason to avoid pushing our current system toward the 400 bar limit?

Fri, Oct 09, 2015

That is a good point, Matt. Thank you for commenting. Yes, that can happen sometimes when buffer salts are present and the organic content is increased too quickly or too much. As you mentioned, flushing with more water and no buffer in the mix should solve this problem.

Thu, Oct 08, 2015

Hi Nancy, Another cause for increasing back-pressure and buffered solutions is buffer precipitation in the column. Although most people remember to flush their columns (or keep a low flow going) it's a really easy question to eliminate at the start. If someone has buffer salts stuck in their column, running a high or 100% aqueous, no buffer, mobile phase through the column has a decent chance of restoring the column to usable condition. Matt