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Derivatization with Dinitrophenylhydrazine


About 0.2 g of frozen plant tissue is homogenized in 20 ml extraction buffer - including the internal standard (standard amount is described in the corresponding analytical method) - with an Ultra Turrax (13000 min -1 ) under streaming argon for 45 s. The extract is shaken for 5 min and then centrifuged at 3200 x g at 4 °C for 10 min. In order to form the 2,4-dinitrophenylhydrazone derivatives of aldehydes, the clear upper phase is stirred for 1 h in the presence of 1 ml of DNPH reagent. The reaction mixture is extracted twice with 5 ml hexane. The collected organic phases are dried under streaming nitrogen. The remaining derivatives are re-dissolved in 0.2 ml acetonitril and stored under argon atmosphere at -20°C until use.

extraction buffer:
methanol/2 mM HCl (1:1, v/v) with 0.001 % (w/v) 2-butyl-6-hydroxy toluene (SIGMA)
DNPH reagent:
0.1 % (w/v) dinitrophenylhydrazine (SIGMA) in 1 M HCl

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Oxylipin Profiling Database All rights reserved 2004 - 2005 A. Schomburg, Plant Biochemistry, University of Goettingen