LC System Flush

[Chris]

We currently run 4 HPLC systems in our lab and each system typically runs 3 to 4 different methods per week so we are continually swapping mobile phases. In order to ensure systems are fully flushed off I would like to introduce a single wash procedure to cover at least 80% of our applications so that there is no confusion as to how systems sholud be washed.

All of our methods use C18 columns and most mobile phases consist of acetate or phosphate buffers with acetonitrile and/or methanol, so I am proposing the following:

20 min on 95% H2O 5% MeCN
20 min on 95% MeCN 5% H20
then 20 min on 50/50 MeCN/H2O and use this as the storage solution

This would also be useful as we are moving towards tertiary gradient systems and it would be useful to avoid swapping the wash solutions round and keep the same wash profile for the majority of methods.

Does anyone employ a similar procedure or envisage this reducing (or increasing) the lifetime of the columns/HPLC systems.

Thanks
Chris

[Uwe Neue]

In our C/U manuals we recommend to store reversed-phase columns in 100% organic, if they are set aside for more than a few days. The reason for this is the prevention of a slow hydrolysis that happens when the column is stored in an aqueous medium. On the other hand, if the column is only stored for a day or two or overnight, I always recommend to store the column in mobile phase, since it takes a long time to do all the washing and reequilibration, if you store the column in something else. With proper caps on the column, there is no issue of a column drying out in a buffer solution over such a short time.

[pipettemonkey]

In our C/U manuals we recommend to store reversed-phase columns in 100% organic, if they are set aside for more than a few days. The reason for this is the prevention of a slow hydrolysis that happens when the column is stored in an aqueous medium.

What organic solvent was typically employed for this storage?

Do you suppose this issue of slow hydrolysis in presence of un-buffered aqueous solvent is much of a threat to modern C-18 columns?

[Uwe Neue]

We store nearly all our columns in acetonitrile, and this is the generally recommended solvent.

How much hydrolysis one gets, depends on the details of the bonding chemistry, but I can not see why it would depend on the type of silica, i.e. whether you have a high-purity silica or a low-purity silica.

[tom_mizukami]

Hi Chris,

For quite a few years now we have used standard startup, shutdown, and instrument setups. Most of our system use quaternary pumps and we set them up in QC and Stability so channel C is in 20% ACN in water and channel D is always 100% ACN.

We then call a shutdown method that rinses buffers from the system using C and then washes and leaves the column in D. Same with startup, rinse with C then use A and B as your method requires.

We always leave our columns in 100% ACN even for overnight storage. Reverse phase mobile phases usually equilibrate quickly. I rather know my column is clean than avoid the 20 CVs to re-equilibration time.

If anyone picks up a column, you know what it is stored in. The only exception is for some columns used with older methods that require ion pairing agents. These are generally stored in mobile phase and we put a sticker on them so they can be easily identified. Good luck!

[Chris]

Thanks Tom, this is similar to what we are looking for, when this was introduced would you say that you noticed any reductions in system or column failures?

[HW Mueller]

As mentioned before, if you have proteins (or some other macromolecules) on the column, you should get them out completely before storing on 100% ACN of MeOH, even when storing only over night.