precautions to take working with a buffered mobile phase

[clairette31]

I would like to work with a buffered mobile phase at pH 8. I think the better is to use Tris-Hcl.
I work with a gradient :
t0 : 10% CH3CN/H20
10 min : 10% CH3CN/H20
40 min : 25% CH3CN/H20
50 min : 100%CH3CN (to eliminate hydrophobic compounds that do not interest me)
60 min : 100% CH3CN
I do semi-preparative purification on an uptisphere ODB 5 column (interchim) 250 *7.8 mm.

Is it a problem to be in pure organic phase at the end ? Salts could perhaps precipitate ? Is it sufficient to wash column with water immediately after he run ?

Thanks for your answers,

Anne-Claire

[Uwe Neue]

1. different buffers have different solubility in the organic medium.
2. you may not need to go to 100% MeCN to wash out your hydrophobic contaminants. Going to only 80% may do the job, without creating a problem with the buffer.
3. If you have a 4-channel system with low pressure mixing, you can get around this completely by briefly washing out the buffer before going to 100% MeCN.

PS: in the previous discussion I pointed out that with the injection mode that you used for prep chromatography, you will get two peaks from the same molecule. Did you follow up on this possibility?

[danko]

Hi Clairette31,

As Uwe wrote above, you may not need 100 % ACN at any time. I don’t know what kind of compounds you’re working with, but take f. ex. proteins; you’re almost certain to get problems with them if 100 % ACN is flushed through the column. And another side effect could be a longer equilibration time after the 100 % ACN.
I have this “philosophyâ€

[Bruce Hamilton]

I've seldom worked with a TRIS buffer on HPLC, so can't comment, but I think Uwe has given you all the necessary advice. There seldom is a need to go to pure acetonitrile to elute material.

The choice of buffer may be determined by the detection method, as well as solubility in the mobile phase.

I'd also try to keep the buffer concentration fairly low to reduce chance of precipitation, and use quality buffer reagents that are classified as suitable for HPLC. I'd try one injection of a sample and go to 90%, and then change the final % to the lowest CH3CN % you can, with a 5 min hold.

I'd also be interested to know if you investigated the solubility issues and the possible injection solvent cause that Uwe suggested. Buffers may slightly mitigate those issues, but probably won't solve them.

Please keep having fun,

Bruce Hamilton