Preparation of Silica Gel for Chromatography ROBERTA H A R R I S AND A R N E N. WICK, Research Laboratories, The Upjohn Company, Kalamazoo, Mich.
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URING the past 2 years the authors have prepared for the chromatography of penicillin considerable quantities of silica gel. This method of preparation is a modification of that reported by Gordon, Martin, and Synge (1) and described in the experimental section of their paper, “Partition Chromatography in the Study of Protein Constituents”. Silica gel which is satisfactory for partition chromatography is difficult to prepare under the best of conditions. Since it is likely to be more widely used in the future, the method of preparation is reported in detail. In a lE-liter, glass-lined tank, 34 liters of soPROCEDURE. dium silicate (Merck’s sodium silicate solution, sp. gr. 1.38 to 1.40,BB. 40” to 42’) and 10 liters of water are mixed with a highspeed stirrer (Lightnin air mixer, Type AR-25, 1750 r.p.m., 0.25 h.p., two 7.6-cm. propellers). Hydrochloric acid (10 N ) is added very slowly (17 liters in 2.5 to 3 hours) with vigorous stirring until the mixture is permanently acid to thymol blue (pH 2.0 to 2.8). 4 large separatory funnel is convenient for the addition of the acid. When about 4 liters of the hydrochloric acid have been added, a very thick, gummy mass is formed. At this point the addition of acid is stopped, and the mass is thoroughly broken up with a heavy wooden paddle. More acid is added dropwise with stirring until a thin suspension results. The rate of the addition of acid can then be increased until the desired pH is reached. The suspension is allowed to digest a t 25” C. with continuous stirring for 2 hours, then filtered by suction on a large stoneware filter (66-cm. diameter). Sharkskin paper (Carl Schleicher and Schull Co., 116 West 14th St., New York, N. Y.) plus a thick
Determination of
filter pad has been used for this filtration. A hard cake of very fine silica gel forms on top of the filtering material. This must be broken up in order to speed the 6ltering process which usually requires about 14 hours. At this point the silica gel can be aged in 0.2 N hydrochloric acid as described by Synge ( 1 ) . This requires 2 days and increases the buffer-adsorbing titer of the product by approximately 20 to 40Y0. I n either case the silica gel is suspended in tap water, filtered, and washed until free of acid. The washed silica gel is dried a t 200” C. for 12 hours and ground to a particle size of 50- to 150mesh, using a Fitzpatrick Model D hammer mill. The ground silica gel is washed -with distilled water until free of chloride (silver nitrate test) and dried in shallow pans a t 250” C. for 24 to 48 hours. This time and temperature are necessary in order t o obtain a product having a maximum buffer-adsorbing titer. Silica gel prepared without aging in 0.2 N hydrochloric acid adsorbs 85 to 100% of its weight of water without becoming lumpy or moist,. For purifying penicillin the less expensive unaged product liae been found satisfactory. The final product is transferred to dry bottles while stili hot and sealed to avoid moisture uptake. From 34 liters of sodium silicate solution approximately 15 kg. of 50- to 150-mesh silica gel are obtained. This is enough for the chromatography oi approximately 100,OOO,OOOunits of Penicillin G. The isolation of pure penicillins using silica gel chromatography will be reported in a separate publication. LITERATURE CITED
(1) Gordon, A. H., Martin, A. J. P., and Synge, R. L. M Biochem. J.,37, 79-86 (1943).
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Amounts of 4-Vinyl-I-cyclohexene (Butadiene Dimer) in Tetrahydrophthalic Anhydride BENJ. WARSHOWSKY AND PHILIP J. E L V I N G Publicker Industries Incorporated, Philadelphia 48, Pa.
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ETRAHTDROPHTHSLIC anhydride (4-cyclohexene-l,2dicarboxylic acid anhydride) is usually prepared by the Diels-Alder reaction ( 1 ) of 1,3-butadiene with maleic anhydride. The product, which is obtained in practically quantitative yield, is pure except for the presence of minute amounts of the dimer of butadiene, 4-vinyl-l-cyclohexene, which is formed at the temperature needed to keep the maleic anhydride liquid for ready reaction--e.g., 80” to 100” C. From the viexvpoint of control and constancy of product, it was desirable to have a rapid, simple method for determining fractions of a per cent of the butadiene dimer in the tetrahydrophthalic anhydride. A solution 15 as found in the selective extraction of the vinylcyclohexene from aqueous alkaline solution by cyclohexane and in the determination of the vinylcyclohexene in the cyclohexane extract by the determination of the extent of unsaturation. Subsequent to the work described in this report a method for the determination of vinylcyclohexene in recycle styrene was described by Laitinen, O’Brien, and Wawzonek ( 2 ) . The sample is dissolved in dioxane and the styrene polymerized on refluxing, using sodium a s catalyst; the vinylcyclohexene left in solution is determined by the iodine chloride method. The method is sensitive to 0.2% vinylcyclohexene and is accurate to +0.2’30 (absolute) over a range of 1 to 2OY0 vinylcyclohexene. Since this method involves removal of the principal constituent by selective polymerization, it could not be applied to the determination of vinylcyclohexene in tetrahydrophthalic anhydride; in addition, the sensitivity was insufficient for the range of concentration covered. 276
PROCEDURE. The sample size ustd is governed by the amount of vinylcyclohexene believed present. For the range of 0.01 to 0.10% a 10-gram sample, weighed to the second decimal place. of product tetrahydrophthalic anhydride is added to a 250-ml separatory funnel containing 40 ml. of aqueous 10% sodium hydroxide solution, followed by the addition of 50 ml. of cyclohexane (free of unsaturated material). After agitation and separation into layers, the lower aqueous layer is removed and the extraction of the cyclohexane fraction is repeated. Three extractions are sufficient. The extent of unsaturation of the cyclohexane extract can be satisfactorily determined by the bromide-bromate titration method of Mulliken and Wakenian ( 3 ) . The percentage of vinylcyclohexene in the sample is readily calculated from thebromine number found, 296 being the bromine number for pure vinylcyclohexene, or from the milliequivalents oi bromine consumed, 27.05 being the equivalent weight of viny!cyclohexene. When the procedure was applied to samples of v i n y l c y ~ ~ i ~ hexene-free tetrahydrophthalic anhydride (bromine number = 105), the cyclohexane extract had a bromine number of 0.00. Application of the procedure to small samples of vinylcyclohexene showed quantitative recovery within 10 relative per cent. Analysis of synthetic mixtures of vinylcyclohexene and tetrahydrophthalic anhydride containing 0.02 to 2.0y0 of the former showed similar recovery. The vinylcyclohexene used in these experiments was obtained by careful fractionation of the dimer prepared by the liquid-phase thermal dimerization of butadiene. The physical constants of the material used checked the values reported in the literature. Analysis of a typical preparation of tetrahydrophthalic anhydride indicated 0.038 * 0.0035?0 vinylcyclohexene to be present
