We use a lot of buffers in our HPLC work. After finishing all the analyses we usually flush the system with water for 25-30 minutes (10 column volumes) to flush out buffers followed by MEOH or ACN to flush out water. Some of the other discussions in the forum mention that the c18 chains can collapse in 100% water. Please advise whether in our procedure of column flushing the buffer ions can get physically trapped and may require longer time for coming out of the column. Perhaps they will be flushed out faster if we use 5 -10% MEOH or ACN as the c18 chains will be straight and will not trap buffer ions. Another question is what is the ideal volume of water required to flush out buffers? Whether 10 column volumes is ideal or too low/high for different kinds of buffers.

With best regards

Dear all,
On a separate question on buffers, is there any way to restore a column which buffer has been left stationary for an overnight run. Its a 250x4.6 c18.

Any Ideas or opinions are appreciated!

Sanjeev,
To answer your question, "they" say that you can adequately flush out your buffer salts by switching your MP composition from buffer to water at the same concentration. IE, if you're running 65% buffer:35% MeOH, just go to 65% water:35% MeOH. I don't know if this truly works, it's just what I hear at seminars and the like. Personally, I always flush my column with 100% water, but it's a C-3 column, so phase collapse isn't a problem.

For the second post, I'd do the same thing: flush with water in place of buffer in the MP and see how things go. I'd probably go to about 5% organic and let it flush there for a while. You can always "restore" a phase-collapsed column by flushing in 100% organic. As long as you ramp slowly to the higher % aqueous, I'd think that you will get most of the buffer salts out before the phase collapses totally.
And I use the 10-column volume rule of thumb too.
Hope this helps!

Is there any advantage/disadvantage for flushing with 100% MeOH or ACN? I know about 100% water and phase collapse, how about 100% organic?

Thanx,
Steve

If you are running buffers and switch immediately to 100% organic, you run the risk of precipitating the salts in your pump or column. I usually flush by switching to the same percent aqueous/organic without buffer as Lisa discusses, then increase the percent organic for storage of the column. For long term storage, a high percentage of organic is needed to prevent the growth of microorganisms. Also, for long term shutdown of you HPLC, flush with 100% methanol, as it is a better "wetter" than acetonitrile.

Another benefit to flushing with 100% organic would be to flush out some of the "junk" that hangs onto the column. Depending on what you inject, this could increase the life of your column substantially.