HPLC problelm

[aussie75]

Hi,
I'm new on this forum! My first language is not english sooo sorry if i make mistakes!
I'm not an HPLC expert and I need your help to solve a problem. I have to determine the title of the Hydroxyproline, using this method :
- Column Perkin Elmer Brownlee validated C18 3micronm 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 result area of standard's analyses are not comparable, not reproducible...in this way I cannot do the areas average and calculate the title of my Hidroxy proline!
Can u help me?
Thank you!!!!!!

[HW Mueller]

It has been a while since I have done this, but off the top of my head:
(Incidentally, your "title" is titre or concentration?)
There are very many descriptions of the FMOC-CL derivatization, look at some, pick what you think is best, jostle around a bit (time, temp. . . .).
You are using adamantyl amine to get rid of excess FMOC-CL? Because of this late eluting entity you use a gradient? I would use glycine or alanine to get rid of excess FMOC-Cl, early, and then be able to use an isocratic method. Also I would have started this with a commercial hydroxyproline-FMOC "standard". (I think just about all amino acids are available as their FMOC derivatives from a company which merged with Merck, Darmstadt??). That way it is easier to pick out the H2O and "NH3" derivatives in your own preps.