Eliana M. Alhadeff, Andrea M. Salgado,

Nei Pereira Jr., and Belkis Valdman[6]

Depabtamento de Engenhabia Qumica, Ehcola de Qumica,
CT/UFRJ, Ilha do Fundao, Cidade Univebhitabia,

CEP 21.949.900, Rio de Janeibo, RJ, Bbahil,

E-mail: belkih@eq. ufrj. bb

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.

Index Entries: Biosensors; gasohol; immobilized enzymes; alcohol oxi­dase; horseradish peroxidase.

Introduction

Since 1975, Brazil has supported a governmental program to design a new car engine technology using (95%) hydrated ethanol as biofuel.

Table 1 Enzymatic Ethanol Biosensors Enzyme Support Transductor Detection range Alcohol dehydrogenase (6) Flat glass Amperometric 0.4-3.9 M ethanol in air AOD and HRP (8) poli(carbamoil)sulfonate — poliethilenimine hydrogel Amperometric 0.02-3.75 mM AODand HRP (4) Not immobilized Spectrophotometric 1.1-21.7 mM AOD and HRP (9) Chitosan Spectrofluorometric 0.01-0.04 mM Alcohol dehydrogenases and NAD+/ NADH (5) Not immobilized Spectrophotometric 0.01-2.5 mM AOD and catalase (10) Controlled pore glass Thermometric 1.9-3.9 M Alcohol dehydrogenase and diaphorase (11) Semiconductor chip X-ray photoelectron spectroscopic 0.005-1.0 mM

Nowadays a worldwide focus is on renewable fuels, and ethanol has been considered an interesting option to replace petroleum derivate gasoline. It has been reported that the United States, France, Switzerland, Austra­lia, Canada, China, Russia, India, South Africa, and the European Com­munity are considering gasoline ethanol blends as fuel options. Recently, Brazilian government funds have been awarded to laboratories in univer­sities and research centers in different regions of the country, and a strict control of the physicochemical characteristics of the gasohol blend and hydrated fuel alcohol for combustion machines is necessary to prevent adulteration (1,2).

Among the physicochemical methodologies developed to identify chemicals, biosensors have been studied in the last 10 yr as analytical instruments that can be applied in clinical, food, and environmental analy­ses. Biosensors to be used as analytical instruments should present some important technical characteristics, such as low response time, high selec­tivity, relatively long lifetime, stability under the analytical conditions, and reproducibility of the measurements (3). Biosensors, which have many advantages, can be miniaturized and/or introduced in on-line pro­ceedings as analytical instruments to detect chemical concentrations with a very rapid response. Recently, increased applications of integrated biosensors and flow injection analysis (FIA) systems in monitoring and controlling biochemical processes have been reported (4-6). Table 1 lists various enzymatic ethanol biosensors published in recent literature. Amperometric, spectrophotometric, thermometric, and X-ray photoelec­tron spectroscopic methods were used to detect ethanol samples using free and immobilized enzyme systems. Alcohol dehydrogenase and NAD+/NADH, alcohol dehydrogenase and diaphorase, alcohol oxidase (AOD) and catalase, or horseradish peroxidase (HRP), have usually been applied in ethanol biosensors, as shown in Table 1.

To improve the quality control process of gasohol and hydrated etha­nol, an automated FIA system was developed using AOD and HRP enzymes, and addition of 4-aminophenazone and phenol. A colorimetric detection method was used in two different methods of analysis, with free (4) and immobilized enzymes. Both systems have shown good results when compared with established methods such as gas chromatography (GC) and high-performance liquid chromatography (HPLC) (4,7).