Equilibrating silica flash chromatography columns is something I always do. There are chemists who see this as an unnecessary, time-and-solvent-wasting step. However, because getting consistent, predictable results is a priority for me, I equilibrate to remove the variability that can be caused by heat generated as solvent initially contacts the silica. Consistency is really important when running flash column chromatography because re-runs are time consuming and may put your compound at risk.
In this post, I examine the role of equilibration speed and duration to show its impact, or lack there of, on purification performance.
When solvent comes in contact dry, active silica, it is adsorbed to the surface creating some friction and displacing energy in the form of heat while increasing backpressure. The amount of heat generated depends on the solvents, their individual heat capacities, and the solvent velocity. Polar organic solvents tend to generate more heat than less polar organic solvents. Faster flow rates create more friction and, therefore more heat than slower flows. I once saw a column get so hot that it melted the outlet frit, ouch!I previously posted on the benefits of equilibration in normal-phase flash chromatography. Besides displacing the entrapped air in the silica’s pores, the equilibration solvent wets the silica and, with enough volume, removes the heat band created by both friction and the heat of adsorption.
So, the solution to these issues has been to equilibrate at slow solvent velocity with copious volumes (3-5 column volumes (CV)), which are the very reasons many chemists prefer to take their chances without equilibrating before purification.
Those of you who use the Biotage® Isolera flash system know that the default equilibration flow rates are the same as the actual method flow rates with durations of 3 or 5 CV, depending on the gradient’s starting solvent percentage. You can override the default equilibration flow rate (I always do) and make it higher to speed the process along. This works but still consumes 3 or 5 CV, however it is MUCH faster.
In reality, equilibration does not need to be slow and consume a lot of solvent. We recently found that we could reduce equilibration volume to 2 CV when increasing flow rate beyond what anyone would deem practical. Yes, pressure and the heat initially spike but the process is so fast that the column is fully equilibrated and ready to use in seconds (with smaller columns). This is a nice development as it addresses the two largest criticisms chemists have of equilibration – time and solvent volume.
So, what solvent velocities are practical, really. Well, some colleagues of mine ran equilibration tests at 100 mL/min for 2 CV using a new flash column, Biotage® Sfär, with 20 µm particles and a 20 bar (290 psi) pressure rating. To do the work they used the new Biotage® Selekt flash chromatography system, with a top-end flow rate of 300 mL/min and maximum pressure of 30 bar (435 psi). A summary of their results is in Table 1.
|Column size (g)||Equilibration volume (mL)||Pressure (bar)||Pressure (psi)|
But I wondered, can I do the same on my Biotage® Isolera Dalton flash-MS system? I typically equilibrate 10 gram Biotage® SNAP Ultra columns (25 µm) at 100 mL/min with no issues it. So, I thought I would try the updated equilibration protocol (150 mL/min!) on it with a 10 gram Sfär HC (20 µm) column. My sample for this test is what I have nicknamed the “Reaction Mix From Hell”. This very inefficient synthesis generated A LOT of by-products and a minimal amount of the target compound. We now use this sample in our new hire flash chromatography training – fun (for me anyway)!
For my experiments, I created a step gradient (11% EtOAc in hexanes for 2 CV followed by 24% EtOAc for 13 CV). Using the 10 gram Sfär HC column, I used the default equilibration flow rate (40 mL/min) and duration (5 CV which equals 75 mL). The equilibration duration is just short of 2 minutes. The resulting purification was as expected, Figure 1.
Figure 1. Reaction Mixture from Hell purification after equilibrating using the default flow rate (40 mL/min) and duration (5 CV).
Then, I used the same gradient method and increased the equilibration flow rate to 150 mL/min, but for only 2 CV (30 mL). This resulted in a 12 second equilibration! Overpressure did not occur, frits did not melt, solvent did not leak, and the results were great, Figure 2.
Figure 2. Purification of the reaction mixture from hell after equilibrating at 150 mL/min for 2 CV shows a nearly identical separation as that generated using the default equilibration.
So, when it comes to silica column equilibration, do it fast, but do it smart. Use columns capable of withstanding elevated equilibration flow rates and keep the duration to 2 CV.
For more information on flash chromatography, I invite you to listen to my webinar on this subject.