Development and Application of an Integrated System for Monitoring Ethanol Content of Fuels

Abstract

An automated flow injection analysis (FIA) system for quantifying ethanol was developed using alcohol oxidase, horseradish peroxidase, 4-amino-phenazone, and phenol. A colorimetric detection method was developed using two different methods of analysis, with free and immobilized enzymes. The system with free enzymes permitted analysis of standard ethanol solution in a range of 0.05-1.0 g of ethanol/L without external dilution, a sampling frequency of 15 analyses/h, and relative SD of 3.5%. A new system was designed consisting of a microreactor with a 0.91-mL internal volume filled with alcohol oxidase immobilized on glass beads and an addition of free peroxidase, adapted in an FIA line, for continued reuse. This integrated biosensor-FIA system is being used for quality control of biofuels, gasohol, and hydrated ethanol. The FIA system integrated with the microreactor showed a calibration curve in the range of 0.05-1.5 g of ethanol/L, and good results were obtained compared with the ethanol content measured by high-performance liquid chromatography and gas chromatography standard methods.

Integrated Biosensor Systems for Ethanol Analysis

“…Alcohol oxidase (AOD, Sigma) and horseradish peroxidase (HRP, Toyobo of Brazil) were immobilized separately on aminopropyl glass beads treated with 2.5% (v/v) glutaraldehyde, as previously described [3]. The composition of the indicator solution was: 4-aminophenazone 0.395 g/l and phenol 0.875 g/l, prepared in a 0.1 M sodium phosphate buffer solution (pH 7), which was also used as carrier [3,13]. Reactions were carried out at room temperature (20°C The resulting coloured product, monoimino-p-benzoquinone-4-phenazone, has been detected with a spectrophotometer at 470 nm.…”

“…Each system had been tested, after calibration, on both response signal repeatability and stability of the immobilized enzymes, when reused in successive analysis. The results were compared to gas chromatography and high-performance liquid chromatography, having shown good agreement [3,4,12].…”

“…Among those, flow injection analysis (FIA) is a very promising technique for being reliable, reproducible, reagent-saving and readily automated. FIA systems have also been applied for ethanol detection either in alcoholic beverages as for gasohol mixtures, using sequential enzymatic microreactors, packed with enzymes such as alcohol oxidase and horseradish peroxidase, immobilized on chitosan or glass beads [2][3][4]. A real alternative to FIA systems is the sequential injection analysis (SIA) technique due to its more versatile sample-handling capability [5].…”

Quality control of gasohol using a micro-unit for membraneless gas diffusion

“…Flow analysis techniques have also been applied in method development for ethanol analysis. Alhadeff et al reported some enzymatic methods using flow-based techniques for determination of ethanol contents in gasohol fuels [8,9] and in fermentation bioprocess [10]. This paper reports the development of a new method for quantitative analysis of ethanol in gasohol.…”

“…In principle, the method in this paper should be more robust than use of the enzymes [8][9][10], and the method has a good potential in further development for making a portable device for quality control of gasohol.…”