An HPLC-based assay for soil enzyme activities using 4-nitrophenyl substrates.

Williams C Iwebema, Joerg Geistlinger, Anita Kirmer, Wilfried Rozhon

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Article in MethodsX, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.

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4 authors.

Williams C IwebemaAnalytical and Bioanalytical Sciences, Department of Agriculture, Ecotrophology, and Landscape Development, Anhalt University of Applied Sciences, Bernburg, Saxony-Anhalt, 06406, Germany.

Joerg GeistlingerAnalytical and Bioanalytical Sciences, Department of Agriculture, Ecotrophology, and Landscape Development, Anhalt University of Applied Sciences, Bernburg, Saxony-Anhalt, 06406, Germany.

Anita KirmerVegetation Science, Department of Agriculture, Ecotrophology, and Landscape Development, Anhalt University of Applied Sciences, Bernburg, Saxony-Anhalt, 06406, Germany.

Wilfried RozhonAnalytical and Bioanalytical Sciences, Department of Agriculture, Ecotrophology, and Landscape Development, Anhalt University of Applied Sciences, Bernburg, Saxony-Anhalt, 06406, Germany.

Funding

No grant is acknowledged in the PubMed record.

6 · The paper itself

Abstract

Soil enzyme activity assays are useful for assessing the biogeochemical potential of soils. Frequently, 4-nitrophenyl-labelled chromogenic substrates are used that yield yellow 4-nitrophenol (4-NP), which can be quantified by spectrophotometry at 410 nm. However, this approach is often impeded by the presence of organic matter, which causes yellow or even brownish colouring of the soil extracts, interfering with spectrophotometric measurements. In this study, we show that high-performance liquid chromatography (HPLC) is an efficient, rapid and selective method for quantifying the liberated 4-NP. The soil samples were mixed with the substrates dissolved in buffers and incubated for a suitable time period. Subsequently, the reactions were analysed by HPLC using a LiChroCART RP18 3 µm 55 × 2 mm with an eluent consisting of 20 mmol L

Indexed as

4-nitrophenyl substratesEnzyme assaysExtracellular enzymesHPLCOrganic matter interferenceOrganic soils

Identifiers

PMID42434050

DOI10.1016/j.mex.2026.104026

PMCPMC13351156

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