i purchased a bondapack amino column of waters make 2 months back

i am analysing only one compound on it with mobile phase of 70 buffer ans 30 acn and flowrate of 1.0 ml/min initially i was getiing a rt at around 30 minutes (buffer is kh2po4 and Nah2po4 . 3.4 gm kh2po4 and 1.97 gm Nah2po4 for 1000 ml of water ph is 6.7 adjusted with 10% Naoh)

after first analysis when i started a second analysis i was getiing rt at 22 minutes (1 months after)


and just now when i have started this analysis i got the rt at 15 minutes with the same chromatographic system so what is the probleam with this column i do not understand

pressure at 1 flow rate is about 350 psi
there is no tremendeous changes in a pressure ofa system initially with column it was about 450 psi

is the column leaching out so regeneration with the h2so4 or nh3 is require

dolan sir please solve my probleam

It appears that your problem could be due to equilibration of amino column. Like Cyano columns, amino columns are usually shipped in organic mobile phase which may not be soluble in H2O. For example, cyano columns that I purchased recently were shipped in 90% heptane and 10% ethyl acetate solvent. I had to completely remove heptane before using it in reversed phase. Try flusing your column with ethyl acetate if it is shipped with mainly non-polar organic solvent.

This is a standard and well understood problem. The amino column is unstable in an aqueous mobile phase, due to the fact that a basic environment is created. It stabilizes by loosing amino groups and creating silanol groups. But this will take some time. During this time, you will constantly loose retention.
The problem could have been avoided, if you would have bought Waters Carbohydrate Analysis Column. It is a bit more expensive than the amino column, but Waters has done the equilibration wash for you already.

Uwe Neue

Uwe,

Do you know what Waters protocole for the (rapid)equilibration of these columns and if yes can you share it with us?