Macromolecules zyxwvut 1980, zyxwvu 13, 19-24 19 Synthesis and Characterization of Poly(glutara1dehyde). A Potential Reagent for Protein Immobilization and Cell Separation Shlomo Margel and Alan Rembaum* Jet Propulsion Laboratory, California Institute zyxwv of Technology, Pasadena, California 91103. Received August 3, 1979 ABSTRACT: The aldol condensation of aqueous glutaraldehyde in the pH range 7-13.5 yielded water-soluble and water-insoluble poly(g1utaraldehyde) (PGL), the molecular weight of which was of the order of 12 to 20000. The structure of PGL was elucidated by means of UV, IR spectrophotometry, electrochemical studies, and the analysis of reaction products of PGL with hydroxylamine hydrochloride. The structure of PGL polymers prepared in a wide pH range was found to be similar. The main differences consisted of changes in the concentration of functional groups. Sufficient evidence was obtained to explain the presence in PGL of the primary hydroxyl and of the carboxyl groups as due to a Cannizzaro reaction. Electrochemical studies confirmed the spectrophotometric evidence of the presence of conjugated aldehyde groups. Recent investigations of water-soluble or -insoluble PGL or of PGL in the form of microspheres indicate that these polymers may yield important immunoreagents for biological research. Glutaraldehyde zyxwvuts has many applications in different areas. It is used as a protein cross-linking agent,' for fixation of living cells2 or t i ~ s u e s , ~ and for sterilization of hospital eq~ipment;~ it was also found to serve as an efficient binding agent of antibodies to micro sphere^.^ Glutaraldehyde was used in a variety of reactions for relatively long periods of time at physiological pH. It was only recently realized that under these conditions a con- siderable amount of glutaraldehyde is polymerized through the aldol condensation mechanism,6 and many reported reactions of glutaraldehyde are actually reactions of glu- taraldehyde as well as of poly(g1utaraldehyde). Although a large number of studies were carried out in the past on the nature of aqueous gl~taraldehyde,~-'' little information is available on the structure of the solid aldol condensation product which was frequently considered to be an impurity associated with glutaraldehyde.12 The present study showed that polymerization of glutaraldehyde under basic conditions results in the production of water-soluble and -insoluble polymers and that these polymers contain nonconjugated aldehyde, conjugated aldehyde, hydroxyl, and carboxyl groups. The weight ratio of the soluble to the insoluble polymer and the concentration of the func- tional groups were found to be dependent on the pH of the polymerization medium. Poly(glutara1dehyde)might be used as a new reagent in protein chemistry and other areas. Only recently the polymer was found to constitute a valuable new reagent for the immobilization of antibodies on solid substrate^.'^ Furthermore, a new method was developed for the prep- aration of poly(glutara1dehyde) in the form of micro- ~pheres.'~ The microspheres were used for cell labeling and cell separation and are suitable also for immobilization of enzymes, drugs, and proteins. Experimental Section (a) Reagents. The following materials were acquired from commercial sources and were used without any further treatment: pyridine, phthalic anhydride, and hydroxylamine hydrochloride (Matheson Coleman and Bell), fluorescein isothiocyanate (Po- lysciences), tetraethylammonium perchlorate (Southwestern Analytical Co.), and ferrofluid (Ferrofluidic Co., Burlington, Mass.). Aqueous glutaraldehyde (Aldrich) was purified by treatment with activated carbon followed by filtration. Di- methylformamide, (DMF), dimethyl sulfoxide (Me2SO) and crotonaldehyde were vacuum distilled. (b) Apparatus. Infrared and UV spectra were obtained with a Fourier transform IR (FTS-l5C, Houston Instruments) and a Cary 219 spectrophotometer (Varian), respectively. Gel permeation chromatography (GPC) was carried out with the high-pressure liquid chromatography Model 6000, fitted with a refractive index detector (Water Associates). The GPC was carried out with dimethylformamide as a solvent and a column of styragel lo5, lo4, lo3 zyxw 8, (pore size); polystyrene was used for calibration. Dupont thermal analyzer 900, Model 950 TGA, was used for thermogravimetric analysis. Polarograms were obtained with a Princeton Applied Research (PAR) Model 174 polarographic analyzer and recorded with a Hewlett Packard Model 7004 zyxw x-y recorder. Cyclic voltammograms were obtained by means of a PAR Model 173 potentiostat driven by a conventional signal generator. The voltammograms were recorded with the x-y recorder. Controlled potential coulometry was studied with the PAR Model 173 potentiostat equipped with a Model 179 digital coulometer. The reference calomel electrode was isolated from the main cell compartment by means of a fritted glass disk. The auxiliary electrode was a platinum wire (diameter 0.076 cm) sealed in soft glass. The working electrode was a hanging mercury drop electrode. (c) Synthesis of PGL. Glutaraldehyde was added to an appropriate deaerated buffer solution and then the mixture was placed on a mechanical shaker for 72 h. The white-yellowish precipitated polymer was filtered, washed with water, and then dried under vacuum at 45 "C. The mother liquor was dialyzed extensively against distilled water and lyophilized in order to obtain the soluble PGL in solid form. The synthesis of PGL microspheres was already described.14 (d) Aldehyde Group Determination. The aldehyde content of PGL was determined from the percent nitrogen of the oxime prepared by the heterogeneous reaction of PGL with aqueous hydroxylamine hydr~chloride.'~~'~ PGL (50 mg) was shaken at room temperature for 24 h with 500 mg of hydroxylamine hy- drochloride. The polymer was then filtered, washed with water, and dried under vacuum at 45 "C. A similar nitrogen content was obtained when the reaction was carried out at 60 "C or at room temperature but at pH 6.0. (e) Carboxy1,Group Determination. The carboxyl content of the polymer in the salt form was determined by ashing the samples and in the acid form by titration of the polymer dissolved in DMF/H20 (1:l) with 0.3 M NaOH. Similar results were ob- tained when the titration was carried out in warm pyridine. The agreement between the ashing method and the titration method was of the order of 15%. (f) Hydroxyl Group Determination. The determination of the hydroxyl content was based on the phthalation method.17-19 Results (a) General. Monoglutaraldehyde polymerizes slowly in an aqueous media of pH as low as 7, and the polym- erization rate increases markedly with the increase of hydroxyl ion and monomer concentration as well as tem- perature.6y20 The rate changes at high dilutions can be monitored by the near UV absorption spectra bearing in mind that side reactions may alter the absorption maxima. Monoglutaraldehyde absorbs strongly at 285 nm (t zy = 4.2 0024-9297/80/2213-0019$01.00/0 0 1980 American Chemical Society