Guest Author: Senior Scientist, Seyed Sadjadi.
Congratulations! You are the proud new owner of a small stainless-steel tube packed with white sand, which will bring you fabulous results…if it lasts its life expectancy.
This is the first in a series of articles to bring to light the life and death of an LC column.
First, let’s look at how an LC column is manufactured.
We start with silica particles (media) of a specific morphology, size, and porosity. The silica particles must also be free of collusion and contaminants, such as metallic and other trace element impurities.
Figure 1 is an SEM image of Kinetex 2.6 µm particles. Notice the perfect spherical shape of all particles.
Once the media is adequately prepared and its surface is ready, it is time to attach the stationary phase to the silica surface. This is a bonding process and the property of the bonded ligand defines the retention mechanism of the column and its subsequent characterization. For example, a reversed phase (RP) chemistry contains bonded hydrophobic ligands, such as a benzene ring, n-butyl, and n-octyl. Similarly, bonded ligands with polar moieties will produce normal phase. And if the ligands contain easily- ionizable and/or ionic moieties then an ion-exchange chemistry is produced. Ultimately, the LC column is referred to by the chemical characteristics of its stationary phase. For instance, a phenyl chemistry is considered as a RP column.
Some of the popular RP stationary phases are depicted in Figure 2 below. From top to bottom, these are Biphenyl, Phenyl, Phenyl-Hexyl, C8 (aka Octyl) and C18 (aka Octadecyl).
Figure 2: 1. Biphenyl, 2. Phenyl, 3. Phenyl-Hexyl, 4. C8, 5. C18
The surface of the silica now contains the main stationary phase and free silanol groups (Si-OH). These groups can help and hinder the retention behavior of the column. On one hand, under RP conditions, the silanols groups can cause peak tailing specifically for the basic compounds. These groups can also help improve the retention and resolution of some compounds. However, a secondary bonding process may be performed to limit these free silanol groups. This process is called end-capping. In most cases, the end-capping agent is a trimethylsilane (TMS). Based on an individual application, the end-capping agent may carry additional polar groups to further enhance the chromatographic characteristics of the column. However, for the case at hand, we will only consider a neutral end-capping agent such as TMS and a C18 stationary phase.
Second, what if we worked in a utopian lab? All plans would execute per their design, all conditions would be perfect, and absolutely NOTHING abnormal would happen. Under this scenario, our column gradually ages and only normal wear and tear is responsible for a column’s demise after many thousand injections of extremely clean samples.
- Loss of end-capping agent
As silica particles gradually lose their end-capping agent, more of the silanol groups are exposed, so their activity increases. This extra activity can be significant for analytes that possess functional groups capable of interacting with the now-free silanols. That means basic compounds (possessing amines and amides) are particularly sensitive to this interaction. The physical manifestation of this process leads to tailing peaks. This phenomenon will gradually reduce the peak height, while the retention time remains unaffected.
- Loss of stationary phase
Hydrolysis of the main stationary phase and its loss directly translates in the loss of analytes’ retention times. Additionally, the column loses the ability to focus the analytes at the head of the column leading to extra peak broadening. Because of the extra band broadening, the peaks gradually lose their height and low detection levels become more difficult. This is especially true for compounds with low detector response.
- Clogged and/or obstructed column
On extremely rare occasions the sample components, such as proteins or protein fragments, may solidify and precipitate inside the column or at the inlet frit. This blockage will lead to increased column backpressure due to the restricted pathway. Eventually, the increase in backpressure will cause the pumps to shut down or lead to spectacular leakages from some of the fittings or disconnected lines. The blockage can also occur from any solids in a sample suspension (high possibility) or silica fragments (very low possibility) that will obstruct a column.
Third (and final), we return to a real life situation. Everything that Murphy’s Law predicted that will ruin a perfectly good day of chromatography WILL happen and sometimes all at once. The samples will be dirty, the mobile phase(s) will contain harsh components, the column is previously abused and poorly taken care of, you name it, it’s going to happen. Under these circumstances, the column’s longevity can be shortened significantly. Figure 3 is a SEM image of a silica taken from an obstructed column after about 100 injections. Notice the foamy materials between the silica particles.
So, it is a fact that an old column loses its ability to adequately retain and resolves analytes and/or compounds of interest.
Hopefully, now it is easier to see the stages of a column’s death!
One way to prevent an early column death is using a guard column. Learn more here.