A rapid, sensitive high-performance liquid chromatography analysis of ammonia and methylamine for nitrogenase assays.
A dansyl chloride precolumn derivatization method has been developed for high-performance liquid chromatography analysis of NH3 and/or CH3NH2 produced by nitrogenase-catalyzed reduction of substrates such as N2 (NH3) or diazirine (NH3, CH3NH2). The dansyl chloride reagent can be used immediately after preparation and is stable at 4 degrees C for 1 month. The derivatization products from NH3 and CH3NH2 are prepared by direct treatment of the assay mixture (30 min of incubation) and are then stable in air and ambient light at room temperature for at least 1 day. They are separated isocratically within several minutes on a mu Bondapak C-18 reversed-phase column (Altex) at 2000-2500 psi using an eluent of 7:7:3 H2O:methanol:acetonitrile and are detected fluorometrically (368 nm excitation; 500 nm emission). The NH3 sensitivity is limited by the background NH3 in the reagents and under practical conditions is ca. 0.02 nmol (20-microliters injection volume). Methylamine sensitivity is an order of magnitude greater. The detector response for either product is linear to at least 2.4 nmol. The method is compared to alternative analytical procedures with respect to sensitivity, convenience, and absence of interferences.
Bravo
M
M
Department of Chemistry, University of Southern California, Los Angeles 90089-0744.
Eran
H
H
Zhang
F X
FX
McKenna
C E
CE
D000641:Ammonia / Q000032:analysis*; D001395:Azotobacter / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D007202:Indicators and Reagents; D008744:Methylamines / Q000032:analysis*; D008832:Microchemistry; D009591:Nitrogenase / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods
D007202:Indicators and Reagents; D008744:Methylamines; D000641:Ammonia; C027451:methylamine; D009591:Nitrogenase