Technical Service "Red Flags" - LC

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 Syringe Filters or Thomson Single Step filter vials, 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 contained in this article "Restek Liquid Chromatography (LC) Columns — 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!