Optimization of the Selectivity of Pyrene Immunoaffinity Columns Prepared by the Sol-Gel Method Margit Cichna and Peter Markl Institute of Analytical Chemistry, University of Vienna, Wa ¨ hringer Strasse 38, A-1090 Vienna, Austria Dietmar Knopp* and Reinhard Niessner Institute of Hydrochemistry and Chemical Balneology, Technical University Munich, Marchioninistrasse 17, D-81377 Munich, Germany Received April 22, 1997. Revised Manuscript Received July 18, 1997 X The sol-gel method opened a new path for synthesizing immunoaffinity column packing materials by encapsulating antibodies in the pores of a silica glass matrix. This paper describes different strategies for the optimization of the selectivity of pyrene immunoaffinity columns synthesized by encapsulating the immunoglobulin (IgG) fraction of an anti-pyrene antiserum in a porous silica glass. The selectivity of the columns which are designed for the determination of pyrene in aqueous samples is limited by the retention of cross-reacting and nonspecifically adsorbed polycyclic aromatic hydrocarbons (PAHs). An optimum in selectivity is achieved by a new method to wash the columns with a mixture of a high molecular weight polymer which blocks nonspecific adsorption sites and a surfactant which removes unwanted substances by inclusion into micelles. Optimal results were achieved by washing with 5% (v/v) Roti-Block in 10:90 acetonitrile-water (v/v). Roti-Block is the trade name for a mixture of a high molecular weight poly(vinylpyrrolidone) with a nonionic surfactant dissolved in PBS buffer. 1. Introduction A large part of analytical chemistry deals with the generation of information on the chemical composition of materials. The data produced are needed to solve problems arising in a wide variety of fieldssfrom material sciences to clinical and environmental chem- istry. An ever-increasing number of these problems demands the determination of trace levels of analytes in complex samples. These problems can be solved by applying analytical procedures that consist in separa- tion methods with high selectivity necessary to elimi- nate interfering matrix components and enrich analyte traces in combination with determination methods allowing the selective determination of the analytes with very low detection limits. Present attempts to solve the problems posed by the determination of nonpolar traces of analytes in aqueous environmental samples mainly use solvent extraction or reversed phase liquid chroma- tography (solid phase extraction) as separation steps. Both methods have disadvantages: solvent extraction is of limited selectivity and can be used to achieve high enrichment factors only if the bulk of the solvent is evaporated. The evaporation step frequently causes systematic errors due to the loss of more volatile analytes. Although solid-phase extraction offers much better selectivity, the technique approaches its limits when analytes and matrix components are of compa- rable polarity. One solution of these problems consists in increasing the selectivity by the use of immuno- affinity columns. Immunoaffinity chromatography uses the highly spe- cific antigen-antibody interactions for the selective separation of antigens from complex mixtures. In this technique antibodies are immobilized in the column packing material. Complementary features of immu- noaffinity chromatography and high-performance liquid chromatography (HPLC) with a sensitive detection system allow their off-line or on-line combination result- ing in analytical methods of superior selectivity and sensitivity. Immunoaffinity columns have been used in a number of papers for water analysis, 1-5 but columns used were prepared by covalently binding the antibodies to the support material. This immobilization technique frequently leads to changes in the conformation of the antibodies resulting in a loss of affinity for the antigen. The columns used in this paper have been prepared by the sol-gel method which is a mild technique to immobilize biomolecules in the pores of a hydrophilic glass matrix. Compared with other immobilization techniques, the sol-gel method offers a number of advantages. 6-8 Antibodies immobilized by this tech- * Correspondence to: PD Dr. D. Knopp, Institut fu ¨ r Wasserchemie und Chemische Balneologie, der Technischen Universita ¨t Mu ¨ nchen, Marchioninistrasse 17, D-81377 Mu ¨ nchen, BRD X Abstract published in Advance ACS Abstracts, September 1, 1997. (1) Rule, G. S.; Mordehai, A. V.; Henion, J. Anal. Chem. 1994, 66, 230. (2) Thomas, D. H.; Beck-Westermeyer, M.; Hage, D. S. Anal. Chem. 1994, 66, 3823. (3) Pichon, V.; Chen, L.; Hennion, M.-C. Anal. Chim. Acta 1995, 311, 429. (4) Pichon, V.; Chen, L.; Durand, N. J. Chromatogr. A 1996, 725, 107. (5) Rollag, J. G.; Beck-Westermeyer, M.; Hage, D. S. Anal. Chem. 1996, 68, 3631. 2640 Chem. Mater. 1997, 9, 2640-2646 S0897-4756(97)00248-2 CCC: $14.00 © 1997 American Chemical Society