Response Surface Methodology for the Optimisation of a 96-Well Microtitre Plate Coagulation Activity Assay

Abstract

In order to assess the impacts of physical and chemical factors on coagulation and flocculation processes, the 1 L jar test has been employed for decades as an effective laboratory procedure. However, it is not suitable for studies involving screening of minute quantities of coagulants isolated from flora and fauna via chromatographic or solubility-based fractionation. The primary objective of this study was to develop and optimise a coagulation activity assay based on 96-well microtiter plates (MTP). The coagulation activity was determined in round bottom-shaped polystyrene MTPs due to a better signal-to-noise ratio. The response surface methodology via central composite design was used to optimise coagulation activity for shaking time (10–70 s), shaking frequency (300–1100 rpm), settling time (20–60 min) and coagulant dosage (0–140 mg/L). Alum was used as standard inorganic coagulant treating a ~ 200 μL, 100 NTU colloidal bentonite solution. MTP-based coagulation activity experiments with optimised parameters as well as conventional jar tests were also carried out with crude Moringa oleifera seed extracts. The coagulation activity was optimum at 40 s shaking time, 700 rpm shaking frequency, 40 min settling time and 70 mg/L alum. A further experiment on turbid river water was conducted using different dosages of crude MO seed extract with the newly developed 96 MTP coagulation activity assay. The optimum dosage was 105 mg/L confirming that the MTP assay can be applied for natural coagulants too. The coagulation activities for alum and M. oleifera in the MTP-based assay displayed a similar pattern.