Enzymatic Determination of Urea in Milk by Sequential Injection with Spectrophotometric and Conductometric Detection

In this work, an analytical system based on the coupling of gas diffusion separation and sequential injection analysis for urea determination in milk is presented. A versatile manifold that could simultaneously be used for either spectrophotometric or conductometric detection was constructed. The sample and urease solution are sequentially aspirated into the holding coil and sent to a thermoreactor, where urea is enzymatically hydrolyzed by urease and converted into ammonium. This stream merges an alkaline solution at a confluence point where ammonia is formed. Ammonia diffuses through a hydrophobic membrane and modifies the bromothymol blue indicator color, when spectrophotometric detection is used, or changes the conductance of a boric acid solution acceptor stream, when conductometric detection is used.This methodology was applied to the determination of urea in 18 milk samples and the results were statistically comparable with those furnished by the enzymatic recommended procedure. The detection limits were 2.6 × 10-4 and 2.8 × 10-5 mol L-1 for conductometric and spectrophotometric detection, respectively. Repeatability (relative standard deviation, RSD) was better than 3.7% and 2.6% for conductometric and spectrophotometric detection, respectively. Keywords: Sequential injection; milk urea determination; urease; gas-diffusion; spectrophotometry; conductimetry