Determination of N-species in soil extracts using microplate techniques

Colourimetric methods for the determination of NO3−, NH4+ and total N in water extracts of soils using 96-well microplate techniques are described. Nitrate was determined by azo dye formation after reduction to NO2− using a solution of hydrazine sulphate. Ammonium in the soil extracts was purified and concentrated by diffusion as NH3 from small volumes (750μL) of extract treated with MgO into a H2SO4 collector using a double-plate, MicroResp™ method and subsequently determined by the Berthelot reaction. For the determination of total N, samples were oxidised with K2S2O8 at 110°C in a 96×1.1mL polytetrafluoroethylene block with a lid that closed individual wells. The oxidised solutions were transferred to standard plates for colourimetric analysis of NO3−. The recovery of N, measured as NO3−, from NH4NO3 and a range of organic-N compounds was >95%. The limits of quantitation of the colourimetic assays were 0.020mgNL−1 for NO3− and 0.051mgNL−1 for NH4+. The methods were tested on water extracts derived from a range of 10 nutrient poor soils from Scotland. There were acceptable linear correlations between the results obtained by established methods. For soil extracts analysed by the microplate method, the relationship for NO3− was 1.03× result from ion chromatography+0.0055 (R2=0.9961); for NH4+ determined by the microplate method, the relationship was 0.9696×result from a discrete analyser−0.0169 (R2=0.9757) and for total N determined by oxidation in the PTFE microplate the relationship was 0.9435×result obtained by combustion+0.0489 (R2=0.9743). Purification of the NH4+ in water extracts from the 10 different soils by the diffusion method did not result in any systematic difference (paired t-test, p=0.05) between measured concentration values determined before and after diffusion.