Chromatography Forum

Dear friends,
I appreciate replies to my last post and were very helpful in solving problems.
Currently I am facing different problem. I am trying to do reverse phase HPLC using C18 analytical column and gradient elution. Buffer A is 5% Acetonitrile 0.18%TFA. Buffer B is 90% acetonitrile 0.15%TFA. Recently, by mistake I stored column in water over weekend. On monday I injected sample - originaly30 microliter volume in TrisHCL pH8 which I diluted in 500 microliter of Buffer A. In that run there was tailing of peak. Also there were powdery deposits in microcentrifuge tube after vaccuum drying of eluted peak collection.I did a blank run (without sample) next time and buffer coming out of column during gradient was clear. However when i did vaccuum drying of aliquot of buffer i found powdery white deposits again in microcentrifuge tube. Whats the reason?
Is there any salt precipitation.
I would appreciate if you guys can help me out?
Amar

It could be that you see the C18 bleed from the column. What is the pH, and what is the column stability at this pH?

That would be interesting to know whether there is any C-18 column out there which can withstand such a high TFA concentration in 90% ACN.
Also, your powder could be due to crud, like Tris, that accumulated on the column?

HWM-please could you explain your post?

I have always wondered about the use of TFA. 0.1% TFA is very commonly used- it has a pH of around 2. The concentration of TFA in the post above is greater but not vastly greater than this. Does anyone have comments on this issue? I think it is much more likely that the endcapping of a column might be lost under these conditions than the C18 ligand-but this gcould be an equal disaster.

Well, I understand that the pH range refers to mobile phases of high aqueous content. My guess would be that ACN neutralizes the H+ much less than does water, it could be quite active in ACN. Also, it might be that TFA itself (general acid catalysis) does something to a greater extent than in water. Actually I am asking, I am not sure (anymore?).

What bleed you get under these conditions depends on a range of different things. First, the ligand: a monofunctionally bonded ligand is less stable than a di- or trifunctionally bonded ligand. Among monofunctionally bonded ligands, there is a big difference between a dimethyl silane and a di-isopropyl silane. The third factor is the ligand density. Since this is complicated, I figured that one might be able to read this best from the C/U manual of the column manufacturer.