Clean, stable baselines are critical to good liquid chromatography, and properly conditioning your LC column is a helpful way to ensure a good baseline. You’ll need to flush out the storage solvent and equilibrate the column to your solvent mixture, and you may need to use an intermediate solvent to ensure complete mixing and flushing.
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Hi, everyone. Thanks for joining us for this Restek Tip. Our topic today is column conditioning. Now, most of the time, the column is not going to be ready to use right out of the box unless the mobile phase you're using exactly matches our storage solvent. The certificate of analysis has the storage solvent listed, and you can view or download the cert from the link that's provided on the inside of the column box. Most of our columns are stored in solvents that are greater than a 50% organic content. If you're using an organic mobile phase that's different from the storage solvent, you want to first flush the column with 10% or 20% less organic content. Do this for about 10 column volumes and then change over to the solvent that you're going to be using for your method.
Now, if you've forgot the calculations for column volumes, you can use one of these two formulas. Note that the constant is different based on whether you're using a fully porous or a superficially porous particle. If you're using buffers in your method, you first want to flush with 10% or 20% less organic content than was used in the storage solvent. This is to make sure that when you introduce the buffer, you don't get any salts precipitating, which will increase the backpressure and ultimately clog the column. After about 10 column volumes, switch over to the mobile phase you're going to be using with the buffer included, and then again flush about 10 column volumes. Watch for the baseline to stabilize in pressure because that lets you know the column is equilibrating. Now, a lot of people like to do one or two conditioning injections before running your samples. That’s a good way to kind of prime the column and get it ready for use in the conditions that you’re going to be using for your method. So once you do these couple of injections you're ready to run the rest of your batch. I hope this cleared up column equilibration for you. Stay tuned for some tips from your friends at Restek.