Maximizing LC Column Lifetime: 3 Tips to Reduce Physical Deterioration
Keeping your chromatography up to snuff means getting down with sample prep and protecting your column!
In my previous blog, Maximizing LC Column Lifetime: 3 Tips to Reduce Chemical Deterioration, I discussed the chemical factors that can reduce column lifetime and performance. With proper usage, many chemical-related column lifetime issues can be significantly reduced or eliminated.
In fact, it’s the physical deterioration of a chromatography column that quickly shortens its lifetime. The physical limitations of column lifetime often are in direct proportion to the sample that is being injected, quality of the sample preparation, and the overall care and maintenance of the LC system. In many cases, once the particulate or contaminant accumulates on the top of the column – the damage is irreversible.
The following three tips will help reduce physical contaminants from impacting your column’s longevity.
1. Follow a routine instrument maintenance schedule
Ensuring that your chromatographic system is in proper working order is the first step to maximize column lifetime. Routine maintenance should include replacement of the rotor seals, replacing the needle, cleaning the solvent paths, and performing a pressure test. (Need a service plan?)
Routine pressure monitoring can be used as an early signal that particulates are accumulating in the flow path or on the column inlet. In extreme cases, method conditions that use aggressive mobile phases can deteriorate pump seals and shed particulates that quickly plug the column inlet.
Less obvious is the influence of a dull needle. A dull needle, either from extended usage or impacting a vial due to incorrect settings, cannot puncture the vial septum cleanly. Depending on the needle design, this can promote coring that allows small pieces of septum to enter the mobile-phase stream.
This gross particulate accumulation can sometimes be removed by back flushing the system and column inlet to dislodge the material. I recommend following the column manufacturer’s guidelines as some columns, due to their frit designs, cannot be subjected to reversing the mobile-phase stream.
2. Follow good laboratory practice and sample preparation
The most common column killer is bacterial contamination. Using proper mobile phase preparation and filtering with a 0.45-µm or 0.2-µm membrane filter prior to use greatly improves column lifetime, which is especially important when using phosphate buffers at neutral pH.
Under the right conditions bacterial contamination can irreversibly foul a column within hours of first use. Once bacteria take hold of the system, it can be very challenging to remove and prevention is always the best approach to avoid such problems.
As a general practice, we recommend routine flushing of aqueous inlet lines, or have the flow stream switched with high percentages of organic solvent to effectively control bacteria growth before it causes an issue.
Accumulation of sample constituents can also cause drastic changes in retention and impact column performance. Following proper sample preparation procedures, such as solid-phase extraction, can greatly reduce chemical and precipitated particulate contamination from depositing onto your LC column.
Phospholipid and protein removal are great examples of how to extend column lifetime. In many cases, the precipitated contaminants irreversibly bind to the sorbent bed making them impossible to remove. Usually, by the time you attempt to wash the column, any proteins that have precipitated on the column inlet have aged, denatured, and possibly even cross-linked, making them impossible to solubilize. Effective sample preparation to remove deleterious sample constituents prior to injection greatly reduces this type of column deterioration.
3. Use a guard column
A more powerful approach to extend column lifetime is to use a guard column rather than a simple pre-column filter. The guard is a small pre-column (like VanGuard guard columns) that serves as a sacrificial sorbent bed that is replaced when problems occur. If the source of your column problem is dust in the mobile phase, or adsorption of contaminants, the guard column will simply take care of them.
For best performance, you should use a guard column that contains exactly the same packing as the analytical column. If you choose a guard column that does not contain the same packing as your analytical column, you can get significant peak distortion. In addition, you cannot predict how well this guard column is protecting your analytical column.
Also, do not use a larger particle size with the belief that it will last longer. This is a common misconception. Larger particle pre-columns can result in a deterioration of the separation due to band-broadening in the pre-column. Therefore, it is always better to match the packing material in the guard column to the packing material in the analytical column. This allows you both the best possible column protection and the least amount of peak distortion.
For these tips on maximizing LC column lifetimes, I have assumed that the column is used for a single assay or in a single procedure. Under these circumstances, the column contamination is fairly predictable.
If your lab uses many different HPLC procedures, I always recommend that you dedicate a single column, including the guard column, to each procedure. This eliminates cross-contamination of the column from one assay to another and makes the column life predictable and controllable.
If your lab does not have any standard assays, this is a moot point. Even under these circumstances, I would keep the guard column and the analytical column together until a deterioration of the performance requires a replacement of the guard column.
- Maximizing LC Column Lifetime: 3 Tips to Reduce Chemical Deterioration
- Explore: Column Protection Products from Waters
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