Sequential injection analysis as a tool for implementation of enzymatic assays in ionic liquids

An approach based on the use of water/1-butyl-3-methylimidazolium tetrafluoroborate ([bmim][BF4]) mixtures in a sequential injection analysis (SIA) system is presented. The rapid and robust procedure developed was used to evaluate horseradish peroxidase activity in [bmim][BF4] and is intended to be a generic tool for enzymatic assays in ionic liquids.The horseradish peroxidase activity tests were based on the implementation of the 4-aminoantipyrine (4AAP)/phenol test in the SIA system, using 1-naphtol as substrate. Small volumes (12 μL) of sample, reagents and enzyme were sequentially aspirated to the holding coil before being sent to the spectrophotometric detector (λ = 510 nm), where a coloured product proportional to the enzyme activity was measured.The results were compared to those obtained when the assay was performed in water/methanol mixtures under the same conditions, to evaluate [bmim][BF4] as an alternative to conventional organic solvents. Comparative evaluation of the enzyme behaviour revealed that the enzyme activity increased significantly when the assay was performed in water/[bmim][BF4] mixtures.The SIA methodology exhibited good repeatability over the full concentration range (R.S.D. < 3.3%, n = 15) studied, produced approximately 1.7 mL of effluent and consumed approximately 36 μL of solutions prepared in water/[bmim][BF4] mixtures for each analytical cycle.