Determination of N-species in soil extracts using microplate techniques

Abstract

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 (750 μL) 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 × 1.1 mL 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 0.020 mg N L⁻¹ for NO₃⁻ and 0.051 mg N L⁻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.

Introduction

The 96-well microplate, developed principally for use as a disposable bioreactor, offers the possibility of N-analysis (NO₂⁻, NO₃⁻, NH₄⁺ and total dissolved N) of soil extracts by adaptation of existing chemistries previously carried out at larger scales, resulting in savings in the amounts of reagents and wastes. Of particular importance among existing methods are those for the determination NO₂⁻ and NO₃⁻ involving azo dye formation [1] and those for NH₄⁺ involving the Berthelot (or indophenol) reaction [2]. The determination of NO₃⁻ by azo dye formation requires prior reduction of NO₃⁻ to NO₂⁻ and this has often been achieved in flow analysis systems with copperised Cd. The use of copperised Cd in microplates poses constraints because the microplate system involves vertically transmitted light for absorbance measurements. Hydrazine can reduce NO₃⁻ to NO₂⁻ and solutions of hydrazine sulphate containing Cu²⁺ as catalyst have been used in automated air-segmented flow systems [3], [4]. A disadvantage of hydrazine in such analysis is that it may partially decompose NO₂⁻ so that simple subtraction of analytical data with and without hydrazine cannot easily be applied to determine NO₃⁻ in the presence of NO₂⁻. However, using an air-segmented flow system under specific conditions of reagent concentration, pH, temperature and time, it is possible to have quantitative recovery of NO₂⁻ and quantitative conversion of NO₃⁻ to NO₂⁻. Kamphake et al. [3] used hydrazine sulphate at a concentration of 190 mg L⁻¹ at 38 °C for approximately 10 min, whereas, Downes [4] used the same concentration of hydrazine sulphate at 33 °C for 7.5 min.

The procedure for the determination of NH₄⁺ in soil extracts using the Berthelot reaction is well suited to adaptation in microplates as the method involves the addition of only two solutions to the sample. However, the reaction is prone to interferences from organic-N compounds and from divalent cations which may exist in soil extracts or natural waters [5], [6], [7], [8], [9]. These interferences can be overcome by separation of the NH₄⁺ by diffusion in the form of NH₃ [10] but microplate methods for this have not been reported. Several methods for the separation of N by diffusion of NH₃ for isotopic analysis exist, such as those involving suspended paper discs soaked in acid [11] and acid traps wrapped in polytetrafluoroethylene membranes [12].

Microplate based methods for the determination of NO₂⁻, NO₃⁻ and NH₄⁺ in soil extracts and water using the Berthelot reaction have been reported but involve reduction of the oxidised forms to NH₄⁺ with Devarda's alloy, transfer of the reduced solution to a fresh well and the use of sulphamic acid to decompose NO₂⁻ [13]. The performance of a number of substituted phenols as alternative to phenol or salicylate in the determination of NH₄⁺ has been studied; 2-phenylphenol proved to be the most promising with regard to freedom from interferences and a method using this reagent has been described for the determination of NH₄⁺ in KCl extracts of soil [8]_.

A knowledge of the total dissolved N (TDN) in soil extracts and waters is important not only in its own right but also because the difference between TDN and the sum of the inorganic N components (NO₂⁻-N, NO₃⁻-N and NH₄⁺-N) allows estimation of the amount of organic N. The quantitation of TDN (simultaneously with total dissolved P) in soil extracts has been achieved by oxidation of the extract with S₂O₈²⁻ in closed tubes heated in an autoclave followed by determination of the NO₃⁻ produced but on-plate oxidation techniques have not been reported [14]. Microplate methods for the direct determination of urea in soil extracts have been reported [15], [16]. Other reported microplate procedures for the determination of N in environmental samples are the determination of NO₃⁻ in waters using brucine [17] and the determination of NO₂⁻ or NO₃⁻ in waters by fluorimetry [18], [19]. Assessments of the capability of microplate methods for the analysis of NO₃⁻, NO₂⁻ and NH₄⁺ in seawater have been made [20], [21] and used for monitoring NO₂⁻, NO₃⁻ and NH₄⁺ in seawater from aquaculture facilities, although, the determination of NO₃⁻ involved off-plate Cd reduction of NO₃⁻ to NO₂⁻ [22].

Our aims were to: (1) optimise conditions for the determination of NO₃⁻ by azo dye formation employing hydrazine sulphate solution as the reducing agent; (2) devise an NH₃ diffusion method for the separation and determination of NH₄⁺ using microplates; (3) devise an oxidation procedure for the determination of TDN in water extracts of soil by a technique compatible with microplate procedures; (4) establish the limit of quantitation (LOQ) for NO₂⁻, NO₃⁻ and NH₄⁺; (5) to test the procedures on water extracts from a range of nutrient-poor Scottish soils.