On-line monitoring of galactoside conjugates and glycerol by ¯ow injection analysis Fe Âlix Ama Ârita Vega a , Carlos G. Nu Ân Äez b , Beate Weigel c , Bernd Hitzmann c , Juan C. Diaz Ricci d,* a Departamento de Bioquõ Âmica y Biologõ Âa Molecular, Facultad de Ciencias, UNV/EHU, Bilbao, Spain b Ca Âtedra de Bioquõ Âmica, Facultad de Medicina, Universidad National de Tucuma Ån (UNT), Argentina c Institut fu Èr Technische Chemie, Universita Èt Hannover, Hannover, Germany d Departamento de Bioquõ Âmica de la Nutricio Ân, Instituto Superior de Investigaciones Biolo Âgicas (CONICET-UNT), Chacabuco 461, 4000-Tucuma Ân, Argentina Received 1 December 1997; received in revised form 15 May 1998; accepted 26 May 1998 Abstract A biosensor attached to a ¯ow injection analysis (FIA) system was developed for the automatic determination of galactoside conjugates and glycerol. The biosensor was based on the enzymatic reaction of galactose oxidase (GalOD) using galactose, raf®nose, lactose and glycerol as substrates. GalOD converts galactoside conjugates to galactohexodialdose conjugates and glycerol to glyceraldehyde with formation of hydrogen peroxide and consumption of oxygen. Variation of dissolved oxygen in the carrier was estimated utilizing an amperometric oxygen probe. The FIA system consisted in a multichannel peristaltic pump, an injection valve and an electronic transducer which were controlled by the CAFCA software. Stability of the enzyme and optimal working condition were investigated. Optimum pH for the immobilized enzymes under these experimental conditions was 7.4 and the enzyme retained 80% of the original activity after two months of use. Studies on the dynamical response of the biosensor showed that the elapsed time between two successive injections could be as short as 120 s without signal deterioration when the ¯ow rate was 2 ml/min and 50 ml of injection volume. Sensitivity of the biosensor was higher for galactose followed by raf®nose, lactose and glycerol. The sensor showed linear response between 0.2 and 2 mM for galactose, 0.5 and 6 mM for raf®nose, 25 and 250 mM for lactose, and 2 and 200 mM for glycerol. # 1998 Published by Elsevier Science B.V. All rights reserved. Keywords: Flow injection analysis; CAFCA; Amperometric biosensor; Dissolved oxygen; Galactose oxidase; Galactose; Glucose; Lactose; Glycerol 1. Introduction Routine analyses in food industry and the on-line control of bioprocesses demand the determination of several compounds in a fast and trustworthy way. Often these measurements are carried out by gas chromatography, high performance liquid chromato- graphy, thin layer chromatography, enzymatic reac- tions or colorimetric determinations, but these analyses are time-consuming and require off-line pro- cedures. The development of ¯ow injection analysis Analytica Chimica Acta 373 (1998) 57±62 *Corresponding author. Fax: +54-81-24-8025; e-mail: juand@unt.edu.ar 0003-2670/98/$19.00 # 1998 Published by Elsevier Science B.V. All rights reserved. PII S0003-2670(98)00389-4