Journal of Chromatography A, 1154 (2007) 473–476 Short communication Rapid microwave assisted esterification method for the analysis of poly-3-hydroxybutyrate in Alcaligenes latus by gas chromatography Aimesther Betancourt a,b , Abdessalem Yezza a , Annamaria Halasz a , Huu Van Tra b , Jalal Hawari a,∗ a Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montr´ eal (Qu´ ebec) H4P 2R2, Canada b Chemistry Department, Universit´ e du Qu´ ebec ` a Montr´ eal, Case Postale 8888, succ. Centre-ville, Montr´ eal (Qu´ ebec) H3C 3P8, Canada Received 12 February 2007; received in revised form 5 April 2007; accepted 10 April 2007 Available online 19 April 2007 Abstract In the present study, we used microwave energy instead of conventional heating to transform poly-3-hydroxybutyrate (PHB) into methyl 3- hydroxybutyrate (Me-3HB) in acidified methanol (H 2 SO 4 , 10%, v/v) mixture in less than 4 min at 10% microwave power. The microwave assisted method was then applied to analyze PHB produced by Alcaligenes latus. The PHB content in the biomass determined using microwave heating was comparable to the amount found by conventional heating. Moreover, the new esterification method was at least 50 times faster than the conventional method, affording a significant saving of time and energy. Crown Copyright © 2007 Published by Elsevier B.V. All rights reserved. Keywords: Microwave heating; Poly-3-hydroxybutyrate; Esterification; Methyl 3-hydroxybutyrate; Alcaligenes latus 1. Introduction Poly-3-hydroxybutyrate (PHB), the simplest and most com- monly known poly-3-hydroxyalkanoate (PHA), is gaining attention as a substitute for petroleum-derived plastic because of its competing thermoplastic properties with recalcitrant poly- mers derived from fossil fuel and above all because of its biodegradability [1]. PHAs are produced by several microorgan- isms, such as Alcaligenes latus as intracellular energy and carbon storage materials. PHA granules are accumulated under unbal- anced growth (nitrogen, phosphorous, sulfur, or magnesium limitation) or balanced growth (without limitation) conditions and in the presence of excess carbon source [2]. Presently, biomass is analyzed for its PHB content accord- ing to the widespread method developed by Braunegg et al. [3]. The method involves hydrolysis and subsequent methanolysis of lyophilized PHB containing biomass followed by gas chro- matography analysis of the 3-hydroxybutyric acid methyl ester (Me-3HB) produced. However, this method is time consuming and requires more than 3 h of heating in a dry block heater. ∗ Corresponding author. Tel.: +1 514 496 6267; fax: +1 514 496 6265. E-mail address: jalal.hawari@nrc.ca (J. Hawari). Several researchers attempted to improve Braunegg method [3] by changing the acid concentration and the dry-biomass weight [4,5] or by changing the acid and the derivatizing agent [6] using the conventional heating method. Recently, microwave technology has received considerable attention as a green process for sample extraction and prepa- ration in analytical chemistry [7–10]. The major advantage of the microwave digestion technique is its high heating efficiency which allows the occurrence of rapid breakdown of the sample matrix [11]. In the present study, a microwave digestion method was developed for the analysis of PHB in bacterial biomass obtained after fermentation of A. latus in sucrose. The effect of several variables, such as heating time, microwave power, and acid concentration on the methanolysis of PHB was investigated to quantify the biopolymer in the microbial biomass. To our knowledge, the use of microwave heating for PHB esterification has not been reported before. 2. Experimental 2.1. Chemicals Poly[(R)-3-hydroxybutyric acid] was purchased from Fluka (Buchs, Switzerland). Methyl(S)-3-hydroxybutyrate (Me-3HB) 0021-9673/$ – see front matter. Crown Copyright © 2007 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2007.04.022