[aussie75]

Hi,
I'm not an HPLC expert and  I need your help to solve a problem. (Sorry for my English!!!! I am Italian!)
 I have to determine the title of the Hydroxyproline, using this method :
- Column Perkin Elmer Brownlee validated C18 3micron 100x4.6mm.
- Mobile phases : (A)CH3CN and (B) sodium acetate buffer solution at pH=4.05
- Pump parameters: step 0  time 5.0 flow 1.50 A:20 B:80 CURVE 0.0
                           step 1  time  30.0 flow 1.50 A:100 B:0 curve 4.0
                           step 2  time 5.0 flow 1.50 A:20 B:80 curve 1.0
I dissolve the standard in a Boric acid buffer solution at pH=8
I derivatize the standard with Fmoc-Cl and Adamantylamina.
I can obtain a good peak but the results areas of standard's analyses   are not reproducible, not comparable, that means that I cannot make the areas average and calculate the title of the Hidroxy proline!!
Can u help me???
 ???Thank u!!!!

[Arkcon]

You get good peaks, but the area's for the standards are not good -- not reproducible, and not scaling proportionally with injection?  There can be a number of problems with your system -- hardware, derivitaization procedure, and just how "good" your separation really is. I'd start by checking your system -- running a 50:50 mix of eluents, and injecting something simple, caffeine is a favorite, and should work for your column, and see if your system is pumping properly, and your injector is working ok, and your detector is ok, and there is no internal leak.

'Tho it seems to me, that you may be asking a lot from your system, dissolving sample at a widely different pH than running pH.  But if that's the method you have, yoiu may have to work with it.

[JGK]

There could also be some buffer precipitation issues when increasing to (or decreasing from) 100% acetonitrile in your gradient system. The buffer precipitation may be causing some system issues.