Proline contamination

[lirunkai]

i am developping a LC method for amino acids using pre-column derivatization with OPA and FMOC. it is basically well working for most AA except for proline. Although i am using good-quality Agilent reagents (borate, OPA and FMOC), I always have a big proline peak in blank run no matter how carefully i prepare the solutions. The contamination level is about 50 uM.

Does anyone have similar experience or have idea in how to fix the issue?

[HW Mueller]

How do you know that peak is proline? What is your blank?
Why do you use both OPA and FMOC-CL?

[lirunkai]

That ghost peak is perfectly overlaid with proline peak and no matter what gradient i used, they had no seperation at all. my blank is is the diluent I used to prepared amino acid standard solutions. i also tried to use biological grade phosphate buffer and water as blank. They are still there. The reason I used both OPA and FMOC is because I found some application notes at Agilent. I did try to use only FMOC derivatazation, the contamination peak is still there.

[Chromatographer2010]

Is your proline peak in the standard injection smaller than the "proline" peak in your blank?

Does changing the concentration of your amino acid standard change the size of the proline peak and all of the other amino acid peaks?

Might want to get some comercially available FMOC-proline to double check everything.