Selectivity problem in validation

[Karvezide]

Hi,
I have tested the pH value of 2.3, but it doesen't work at all.
Although the change of MeOH from originally of 10% to 5% made some progress, the problem was not completely solved and the analytic time was too long (about 23 min for the internal standard to elute). In this case, a gradient from 5% to 15%, 1%/min MeOH could not complete the whole run in 15 minutes.
The replacement of MeOH by ACN didn' work, too.

Is it meaningful to buy a new column, exactly the same as the publications? One publiction used Alltech Alltima RP-column, the author used perchloric acid to deproteinate and no internal standard was given so that the analysis time was as short as 4 minutes. The other used SymmetryShield RP18, but ACN for deproteination. Internal standard was given, and the analysis time needed 15 minutes. In your opinions, which publication would you like to prefer??

By the way, we have two columns from vydac, one is TP and the other MS. I thought these both are for the polypeptide and protein analyzes (???). We also have a column from Merck Chromolith C18 100*4.6 mm. The iothalamate analyze using this column showes about the comparable chromatogramm as the vadyc columns, but with shorter analyze time.

Thanks a lot again

best regards

[grzesiek]

"In this case, a gradient from 5% to 15%, 1%/min MeOH could not complete the whole run in 15 minutes. " - is there a reason why not to use steeper gradient and shorter column? do you get enough resolution? do you know where did interfering peak come from?

"Is it meaningful to buy a new column, exactly the same as the publications?" - no, publications can help you to develop your own method

"In your opinions, which publication would you like to prefer??" - for what? do you see the need for using internal standard? or can you live without it?