|Amino acids analysis of axis and cotyledons of Pisum sativum by HPLC - Materials and Methods|
Derivatization of individual amino acids with PITC
PITC reacts with primary and secondary amines, leading to PTC-amino acids detectable at 254 nm (see figure below).
This method is sensitive down to at least 10 pmol (Bergman et al.).
Reference : Bergman, T., Carlquist, M., and Jörnvall, H. (1986) in Advanced Methods in Protein Microsequence Analysis (Wittmann - Liebold, B., Salnikov, J., and Erdmann, V. A., Eds.) 45 - 55, Berlin/Heidelberg
Extraction of amino acids from cotyledons and axis and derivatization with PITC
Cotyledons and axis were separated from seeds taken at various times of germination and amino acids were extracted from 20 to 30 organs.
In the case of the dried seed (i.e., 0 hour of imbibition) the whole seed was treated.
Reverse phase-HPLC was performed using a Waters system (Millipore) with :
System control and data processing were performed using Millenium 32 chromatography manager software (Waters).
Derivatized amino acids were loaded on a reverse-phase C18 Nova-Pak column 4 Ám (3.9 x 300 mm) thermostated at 37°C.
Click to see the complete system.
Derivatized amino acids were separated using two mobile phases :
Click to see the separation of the 20 PTC - amino acids
|time (min)||% A||% B||increase of B|