BIOTECHNOLOGY TECHNIQUES Volume 7 No.1 (January 1993) pp.1-4 Received as revised 17th September COLOR]METRIC ASSAYS FOR THE DETERMINATION OF DIOCTYL SODIUM SULPHOSUCCINATE AND GLYCEROL IN REVERSED MICELLES D.M.F. Prazeres 1, F.A.P. Garcia2 and J.M.S. Cabral 1. 1Laborat6rio de Engenharia Bioqufmica Instituto Superior Trcnico 1000 Lisboa, Portugal 2Departarnento de Engenharia Qufmica Universidade de Coimbra 3000 Coimbra, Portugal SUMMARY Two simple and reproducible colorimetric determinations are proposed for quantifying the surfactant dioctyl sodium sulphosuccinate (AOT) and glycerol in reversed micellar media. INTRODUCTION Recently considerable attention has been paid to reversed micelles from both experimental and theoretical viewpoints in different fields (physical chemistry, colloid science, enzymology, protein purification, etc) with a special emphasis in the dioctyl sodium sulphosuccinate (AOT)/isooctane system. Good, simple and reliable methods are needed to quantify surfactant and other constituents in these systems. During the course of investigations on lipase hydrolysis of triolein in AOT/isooctane reversed micelles using the Lowry and Tinsley (1976) colorimetric procedure for determining oleic acid, interference by AOT was noted (Rocha and Garcia, 1992; Prazeres et al, 1992). Further studies indicated that the same colorimetric reagent could be used in the quantitative determination of AOT with good results. The interference by AOT in the determination of fatty acids led us to search for a different way of following the hydrolysis reaction. Instead of quantifying free fatty acids as usual in non- specific lipase hydrolysis, a technique for determining glycerol and AOT was developed. MATERIALS AND METHODS Chemicals and Lipase. Dioctyl sodium sulphosuccinate (AOT) 99% purity from SIGMA was used without further purification. Riedel deHaen supplied isooctane (> 99%) and toluene (> 99%). Glycerol (87 %) and oleic acid (> 99%) were from Merck and triolein (65 %) was from BDH. A mixture of two Chromobacterium viscosum lipases (3880 U/mg protein) from