Tips on equilibration and storage of flash cartridges and prep HPLC columns

Proper equilibration and storage of your cartridges and HPLC columns might sound painful, but believe me, do the steps correctly and you will save yourself a lot of pain in the long run. In this post, learn how to properly equilibrate and store flash chromatography cartridges and prep HPLC columns packed with silica or C18.

This spring literally put a spring into my step and marked another first in my life. After years and years of putting it off, I decided to go jogging! I really liked it and started going every day, but there was just this one little problem. I did not do any type of warm-up before I ran or any type of wind-down activity afterwards. I just burdened my muscles without any care. It comes as no surprise that I ended up jogging my way straight into the orthopedist office with a foot injury.

If you are currently wearing your lab coat rather than your track pants, well dwell on this. Whenever you skip your preparation and wrap-up activities in the chromatography lab, you might not damage your body, but you damage your body of work.

Let us consider flash chromatography cartridges and prep HPLC columns . Before you put them into real purification action, you got to get them ready by equilibration. After you are done the purification you must store them properly to avoid any damage. Skip any of these steps and your columns, cartridges and purifications might suffer irreparable injury.

Before I get into more details on helping you prevent this painful situation, I would like to point out that there are a few important differences between flash cartridges and prep HPLC columns.

flash cartridges, prep HPLC columns, flash chromatography, preparative chromatography
 Flash cartridgesPrep HPLC columns
Particle size15-60µm5-15µm
Pressure during useModerateHigh
HardwarePolymers (plastic)Stainless steel
Type of packingDrySlurry
Packing materialFrequently Silica;
Frequently C18
Frequently Silica;
Frequently C18

Now that you are aware of some of the characteristics of flash cartridges and prep HPLC columns, let us see how we can equilibrate them.

Equilibration when using silica phase as packing material

When cartridges are dry packed with silica, it is important that you first wet the packing bed before you start the real equilibration. This wetting step can generate heat if you are using polar solvents. It is very important that you carefully select the wetting solvent and that you keep the solvent flow moderate especially if you are using bigger cartridges (more silica=more heat). If the generated heath is excessive, you might end up permanently damaging the cartridge.

Once you’ve wet the packing bed and eliminated the heat, there are no further restrictions on solvent choice and flow. Of course, you should always stay in line with material specifications.

If you are using slurry packed prep HPLC columns, congratulations. The bed is already wetted, so you can skip all I wrote above and get into equilibrating the columns immediately.

Typically, equilibration of a column or cartridge is done with 5 – 8 column volumes (CV), usually with the starting solvent mixture of the gradient.

If you are interested in learning more about the packing process, check out my previous posts on dry packing and slurry packing of columns.

Equilibration when using C18 phase as packing material

Most of the C18 phases cannot be equilibrated with 100% water, as this type of solvent leads to phase collapse and permanent damage of the phase. Instead, I would recommend that you add at least 5% organic solvent to your water to avoid this effect.

For best performance during use of your chromatography system , I suggest that as your final step, you equilibrate the column with the starting solvents you will use during the separation.

Storage of consumables packed with silica phase

Silica is a polar material that is very quickly deactivated if it comes into contact with polar solvents. Minimal water traces, that could be present in used solvents or injected samples, can deactivate the material.

For this reason, I would NOT recommend storing plain silica.

Storage of consumables packed with C18 phase

As the C18 phase is bonded and sometimes end-capped, the risk of deactivation is substantially reduced. This gives you the opportunity to reuse the material. To store C18 packed columns, you can typically use a mixture of water and organic solvent 20/80. Organic solvents that are often used for storage include acetonitrile, ethanol and methanol.

To eliminate the risk of phase collapse, you should avoid using 100% water.

For all Silica based phases, regardless if bonded or not, you should maintain the pH within 2-8. At higher pH levels, silica starts to dissolve, causing permanent damage to the phase.

This wasn’t a marathon of a post, now was it? Just some fast, quick and dirty tips you can take and run with so that you can save yourself some troubles in the long run. And if you are looking for some nice ideas on how to keep your columns in top shape during the actual chromatography run, check out my previous post on column maintenance here .

We will also be hosting a free webinar on chromatography consumables and sample loading on May 26th, register for it here:

This is it now with all the running for today, let us not forget to wind down, stretch out, relax, and give yourself a little mental break before you come back to read some posts again!

Till next time,

The Signature of Bart Denoulet at Bart's Blog