Chromatography Forum

Hi everyone,
I am new to this forum and this is my first post! I am a Phd student and have been recommended this forum by my supervisor!
Alright, here is my question.. How can I explain that with a not-so old column I have a well working method, but if I put a brand new column instead (same model), I have slightly different results.. e.g. some unwanted co-elution, or some peak shifting.
Thanks for any help!

Columns’ properties vary more or less even within the same brand etc. Good brands less, not so good brands more.
Under method validation for instance people used to buy the so called “validation column kit” many vendors provide, in order to assess the variation that can be contributed to the column.
These kits consist (typically) of two columns from the same batch and one from another/different batch/lot.
The age of the column has a role to play as well end you’ll often see tailing increase and/or plate count decrease the more you use the column.

Best Regards

An important factor in the separating characteristics of a column is the stationary phase. The usage of the column can change the stationary phase by leaving stuff on the column that changes the phase chararacteristics or by degrading the stationary phase that is already present. Stuff left on the column can do things like hide activity that interferes with a particular separation or change the nature of the stationary phase entirely.

I've seen a packed column GC method that required several injections of yesterdays samples to condition the column before work could begin today - just to leave stuff on the column in order to obtain the separation necesary to run the method. (In that case the stuff eluted off the column in several hours.) LC columns are subject to the same kinds of effects.

Another important consideration is how the column has been treated in use. Has it been allowed to go dry? Has it been dropped? Have samples been diluted sufficiently and filtered prior to injection? It's been my experience that some labs handle their columns much better than others.

Thank you for those answers! This does really help understanding what might have happened.

To answer JMcK, I didn't let the column dry as I was using it continuously on the HPLC. Otherwise I think I took good care of my column i.e. no droppings; and my samples were finely filtered prior to any injection.

Is that possible then, that the salts contained in my first samples have somehow changed the stationary phase of my column, allowing better separation for the following samples?

Thanks again!

Pretty wide open question. It depends on a number of things, including the salts, pH, the stationary phase and the support. The sample matrix may have something in it that has an effect as well. (And sometimes a new column is not just right.)

An older column will have been cycled numerous times, call it extended conditioning if you like, which will affect the stationary phase

Johnny Rood is right. And if it's not actually your personal column that no one else has touched, what has anyone else done with it? For example, if anyone has ever even thought about doing an ion-pair method on it, it will never have quite the same retention times again (because the ion pair reagent will linger, almost indefinitely).

It is my personal column, with a C18 stationary phase. Nobody else (as far as I know), as ever used it before. Only myself and with more or less the same method.
Once the stationary phase has been altered/modified, is it possible to get it back to normal? By conditionning the column for example? Or is it simply possible to prevent that from happenning by cleanning the column regularly?

Thanks again eveyone!

Hi Asmodee,
In my personal experience (I use C18 columns a lot on an LCMS/MS system) it’s quite tricky to actually ‘bring back’ a column which has already started degrading. The best thing you can do is try running your elution solvent 100% at normal flow for a while (perhaps an hour) but I’ve found that when it comes to looking after your columns, prevention is much better than cure. To that end I recommend that each time you finish your run of samples do a ‘blank’ injection with a ‘clean up’ method (i.e. do not actually inject anything, just have the instrument run the method). Our typical cleanup method includes a ramp up to 100% solvent left isocratic at 100% for half an hour. Ideally the solvent shouldn’t contain buffer if performing a cleanup. Also, I recommend not leaving your column with a mobile phase at the extremes of its pH tolerance inside it when the instrument is not running your samples and that you do not leave your column at elevated temperatures (if using a temperature controlled column compartment) when it’s not in use either.
I hope that’s of some help. The above works fairly well for me but I’m sure some more experienced hands can provide further guidance!

Cheers,