Anal. Chem. 1982, 5 4 , 1449-1450 MacAdam, D. L. J. Opt. SOC.Am. 1942, 32,247. MacAdam, D. L. J. Opt. SOC.Am. 1943, 32, 675. MacAdam, D. L. J. Opf. SOC.Am. 1943, 33, 18. Reilley, C. N.; Fiaschka, H. A.; Laurent, S.;Laurent, B. Anal. Chem. 1960, 32, 1218. Reilley, C. N.; Smith, tillls M. Anal. Chem. 1960, 32, 1233. Vytras, K.; Kotrly, S.;Vondriskova, E.; Vorac, B. Collect. Czech. Chem. Commun . 1970, 35 3379. Kotrly, S.;Vitras, K. Talanta 1971, 78, 253. Kotrly, S.; Vytras, K. S6. VedPr ., Vys. Sk. Chemickotechnol. Pardublce 1989, 79, 21. Kotrly, S.;Vltras, K.; Oharek, J.; Vondrusko'va, E. S b . Ved Pr., Vys. Sk. Chemickotechnol. Pardublce 1970, 2Zm19.
1449
(17) Vitras, K.; Vytrasova, J.; Kotrly, S. Talanta 1975, 22, 529. (18) Bhuchar, V. M.; Das, S.R. J. Opt. SOC.Am. 1964, 5 4 , 817. (19) Bhuchar, V. M.; Kukreja, V. P.; Das. S. R. Anal. Chem. 1971, 43, 1847. (20) Cacho, J.; Nerln, C.Afinidad 1981, 374, 345. (21) Cacho, J.; Nerin, C.; Calvo, A.; Ruberte, L. R . SOC. E s p . Fis. Quim. Reun. Bienal, 78th 1980. (22) Nerln, C.; Cacho, J. R . SOC. Fls. Quim. Reun. Bienal, 78th 1980. (23) Goerdeler, J.; Galinke, J. Chem. Ber. 1980, 9 3 , 397.
~
RECEIVED for review December 21,1981. Accepted March 22, 1982.
Colorimetric Determination of 2,3-Butanediol R. A. Speckman' and E. B. Collins" Department of Food Science and TechnologJv, University of California, Davis. California 956 16
2,3-Butanediol (2,3-butylene glycol) is a metabolic end product of many microorganisms. Current analyses involve either cleavage of the diol to acetaldehyde ( I ) or oxidation of the diol to acetoin mid diacetyl followed by determination of the amount of oxidant ( 2 )or products (3). Both methods involve reactions that are extremely susceptible to slight changes in experimental conditions and difficult to control. A method we devised depends on strict adherence to a rigid experimental protocol ithat must be performed in an exhaust hood in the dark and requires red light-resistant tubes, rubber septa, addition of reagent with a syringe, and precise control of pH, heating, and reaction time ( 4 ) . The purpose of this study was t o develop a simple colorimetric procedure for quantitative determination of the microgram amounts of 2,3-butanediol €ound in biological test Bystems.
EXPERIMENTAL SECTION 2,3-Butanediolwas obtained from K & K Laboratories,Jamaica, NY. It was distilled and the fraction at 181 "C was collected and used for preparing standrud solutions. All other organic chemicals were obtained from Aldrich Chemicals, Milwaukee, WI. All inorganic chemicals weire reagent grade. Spectrophotometric measurements were made with a Beckman spectrophometer, Model DB. The procedure we developed for determining 2,3-butanediol requires the following reagents: ethylene glycol, 0.1 M H,I06, 4% (w/v) CuS04, a standard solution of 2,3-butanediol (10 yg/mL), and 1.5% (w/v) p-hydroxydiphenyl in 0.5% NaOH. The p hydroxydiphenyl solutioin should be stored in a brown bottle and is stable up to 1month ,st room temperature. For determining the diol, pipet 1.0 mL of the unknown, adjusted to pH 7.0 (5) and containing 0 to 10 yg 2,3-butanediol/mL, into a test tube and add 4 mL of water. Run a reagent blank and several 2,3-butanediol standards (0-10 yg/mL) parallel. Add 1.0 mL of the periodic acid solution to each test tube, mix, and let stand at rmm temperature for 30 min. Add 2 drops of ethylene glycol to each tube, mix well, and let stand for 5 min. 14dd 0.05 mL of the CuS04solution, mix, add 0.1 mL of the p-hydroxydiphenyl reagent, mix, and incubate for 30 min at 30 "C in a thermostatically controlled water bath. Shake the tubes occasionally during the incubation. Place the tubes in a bath of boiling water for 90 s to destroy the excess p-hydroxydiphenyl and then cool them to room temperature. Determine absorbancy ai, 570 nm using water passed through the periodate reaction as the internal standard. Compare the absorbancy values for unknowns to the standard curve. RESULT13 AND DISCUSSION Investigations with Vanadium Pentaoxide. Diols from which ketones can be formed are cleaved readily by vanadium Present address: Department of Food !3cience, University of Illinois, Urbana, IL 61801. 0003-2700/82/0354-1449$01.25/0
(6), but 2,3-butanediol is oxidized to diacetyl (7), a diketone that can be determined readily with the Westerfeld procedure (8). Solutions (0.01-0.30 M) of vanadium pentaoxide (V205), a stable powder that can be stored indefinitely and used as needed, were prepared in 1 M H2S04(7) and used for testing solutions of 2,3-butanadiol (0-1000 hg/mL). Following oxidation of the diol, with heating from 1 to 30 min in a bath of boiling water, we attempted to test for diacetyl with the Westerfeld procedure. A white precipitate formed upon addition of the a-naphthol, a problem we had encountered earlier in attempts to analyze acidic column effluents. Oxidized solutions of the diol were adjusted to neutrality by dropwise addition of 40% NaOH, and analyses again were attempted. Upon addition of the a-naphthol, the color of solutions became murky brown. We observed that solutions containing the larger amounts of 2,3-butanediol became somewhat green during heating and assumed that the green color resulted from formation of a mixture of pervanadyl ions (yellow) and vanadyl ions (blue). Nevertheless, measurements of absorbancy a t 200-700 nm failed to give a linear relationship between green color and original concentration of 2,3-butanediol. Cleavage of 2,3-Butanediol to Acetaldehyde with Periodic Acid. Periodic acid cleaves stoichiometrically many organic compounds containing vicinal hydroxyl groups, carbonyl groups, or either of these and an amino group (6). Desnuelle and Naudet ( I ) used periodic acid to cleave 2,3butanediol to acetaldehyde. Analysis of the acetaldehyde, however, depends on spectrophotometric measurement of maximal absorbancy of a transient blue color that appears during the reaction, and the method is useful for determining only large amounts of the diol (50-500 yg). Nevertheless, we found their method ( I ) to yield a stoichiometric relationship between 2,3-butanediol and the acetaldehyde produced. Choice of Reagent for Determining Acetaldehyde. The procedure for determining lactic acid by Barker and Summerson (9) actually determines acetaldehyde, employs p hydroxydiphenyl (PP) as reagent, and is very sensitive (0-10 yg/mL). The method of Sawicki et al. (IO),adapted for use in milk systems by Lindsay and Day ( I I ) , employs 3methyl-2-benzothiazolone hydrazone (MBT), which reacts with acetaldehyde in acidic aqueous solutions to give a blue cationic dye. N-Hydroxybenzenesulfonamide (HBS) was used in a method by Ismail and Wolford (12) to measure acetaldehyde in orange juice and dates. We tested and found each of these reagents and methods satisfactory for determining known concentrations of acetaldehyde (0-50 yg/mL), but none gave satisfactory results when used to measure 0-50 yg of acetaldehyde/mL derived by cleavage of 2,3-butanediol with 0 1982 American Chemical Society
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Anal. Chem. 1982, 5 4 , 1450-1452
periodic acid. On the basis of the assumption that periodic acid interfered with detection of acetaldehyde with the reagents, reaction mixtures after cleavage were neutralized with 40% NaOH and treated with arsenite, but neither procedure was successful. Finally, we destroyed the excess periodate in tests by adding 2 drops of ethylene glycol. This procedure was successful with the method of Barker and Summerson (9),which employs p-hydroxydiphenol as reagent, but it was unsatisfactory with the other methods. Possibly the formaldehyde formed upon addition of ethylene glycol interfered with determinations of acetaldehyde employing 3-methyl-2-benzothiazolone hydrozone or N-hydroxybenzenesulfonamide as reagent. Interfering Materials. Barker and Summerson (9) noted that glucose interfers with the analysis, an important point that is not included in two monographs on methods for analyzing fermentation mixtures (5,13). Acetoin, the precursor of 2,3-butanediol (14),would be cleaved to acetaldehyde and acetic acid. Acetoin can be determined separately, and corrections can be made. Diacetyl would yield two molecules of acetic acid and not interfere. Threonine would yield acetaldehyde. Some microorganisms produce acetaldehyde simultaneously with 2,3-butanediol (15). We recommend separation of 2,3-butanediol from acetoin and other interfering
compounds by salting-out chromatography ( 4 ) prior to determination of 2,3-butanediol.
LITERATURE CITED Desnuelle, P.; Naudet, M. Bull. SOC.Chlm. Fr. 1945, 72, 871-875. Johnson, M. J. Ind. Eng. Chem., Anal. Ed. 1944, 76, 626-627. Happold, F. C.; Spencer, C. P. Biochim. Biophys. Acta 1952, 8 , 18. Speckman, R. A.; Collins, E. B. Anal. Biochem. 1968, 22, 154-160. Nelsh, A. C. "Analytical Methods for Bacterial Fermentations": Nelsh, A. C., Ed.; Prarie Regional Lab.: Saskatoon, Canada, 1950. Stewart, R. "Oxidation Mechanisms"; W. A. Benjamin: New York. 1964. West, D. M.; Skoog, D. A. J. Am. Chem. SOC.1960, 82,280. Westerfeld, W. W. J. Bioi. Chem. 1945, 761, 495-502. Barker, J. 6.; Summerson, W. H. J . Bo/. Chem. 1941, 738, 535-554. Sawlcki, E.: Hauser. T. R.; Stanley. T. W.; Elbert. W. Anal. Chem. 1961, 33, 93. Lindsay, R. C.: Day, E. A. J. Dairy Sci. 1965, 4 8 , 665-669. Ismall, M. A.; Wolford, R. W. J. Food Sci. 1970, 35, 300-301. Chayken, S. "Biochemistry Laboratory Techniques"; Wiiey: New York, 1966: DO 89-91. rr - JuAlE.; Heym, G. A. J. &Cter/O/. 1956, 7 7 , 425-432. Wood, W. A. "The Bacteria"; Academic Press: New York, 1961; Vol. 2, pp 59-150.
RECEIVED for review February 12, 1982. Accepted April 8, 1982. This investigation was supported by a grant from the Dairy Council of California.
Determination of Trace Amounts of Sulfur in Organic Solvents Erik Klssa Jackson Laboratory-Chemicals
and Pigments Department, E.
I. du Pont
Sulfur is a known poison of precious metal hydrogenation catalysts. The detection of sulfur in organic liquids to be hydrogenated is therefore of practical interest. Trace amounts of sulfur have been determined by reduction with h e y nickel to nickel sulfide. An addition of acid liberates hydrogen sulfide which is titrated with mercuric acetate (1) or determined spectrophotometrically after conversion with N,N-dimethyl-p-phenylenediamine to methylene blue (2). The elapsed time of the analysis is 3-4 h and the accuracy is only fair. Microcoulometric methods for the determination of sulfur after reduction or oxidation (3-6) are rapid but not applicable to samples containing large amounts of nitrogen or halides. Drushel (7) described a method for the determination of trace sulfur in petroleum fractions involving noncatalytic hydrogenolysis to form hydrogen sulfide determined by the Houston-Atlas H2S analyzer. It was of interest to find out whether the method is applicable to liquids other than hydrocarbons and to sulfur compounds not investigated by Drushel.
EXPERIMENTAL SECTION Apparatus. A Houston Atlas Model 856 total sulfur hydrogenator with a ceramic tube was used for hydrogenolysis. The furnace temperature (center zone) was 1300 "C, turned down to 900 "C when the instrument was not in use. The hydrogenolysis tube was positioned so that its end protruded 70 mm from the furnace (measured from the end wall of the furnace to the end of the "Teflon" TFE fluorocarbon resin union holding the septum). A Model 1002 Micro-jector syringe drive (Houston-Atlas, Inc.) was equipped with a 1OO-pLor 10-pL Precision Sampling Co. CGV syringe. The exit end of the hydrogenolysis tube was originally connected to a microfiber filter (Balston, Inc., Catalog No. DFU 9933-05, grade Q, size in. X 11/4in.) with a Teflon tubing. Since carbon deposits in the tubing restricted flow, we connected the filter directly to the hydrogenolysis tube by means of a Teflon 0003-2700/82/0354-1450$0 1.25/0
de Nemours and Company, Wilmington, Delaware 19898
union. The filtered gases were passed through a gas wash bottle containing 5% acetic acid to the sample chamber of the hydrogen sulfide analyzer (Houston-Atlas, Inc., Model 825R-d). Chemicals. The hydrogen was obtained from Matheson Co., ultrahigh purity grade. The sulfur compounds,obtained either from Eastman Kodak Co. or Aldrich Chemical Co., were analyzed by the Schoniger method for sulfur. Diphenyl sulfone was purified by sublimation. Procedure. Before the furnace temperature was raised to the operating temperature of 1300 "C, the hydrogenolysis tube was cleaned by heating both of its ends which normally protrude from the furnace. With helium flowing through the tube, the exit end was disconnected from the filter and the entrance end pushed into the furnace. After 2-3 min, the exit end of the tube was pulled into the furnace and heated until smoke was no longer visible. The tube was then placed into its operating position and the filter connected to its exit. We found this procedure to be sufficient. For the removal of refractory coke deposits, both ends of the hydrogenolysis tube may have to be opened to allow air to enter the tube and oxidize the accumulated coke (7). While the furnace was heated to the operating temperature, helium was replaced with hydrogen (flow rate 400 mL/min). The syringe was filled with the sample and positioned on the syringe drive so that its barrel was flush with the septum. The syringe drive was operated at speeds 0.25-4.43 pL/min with a 10or 100-pL syringe. When the signal recorded approached its maximum value, the lead acetate paper drive was advanced and the signal recorded again. The second recording representing a steady state was used for the calculation.
RESULTS AND DISCUSSION The Houston-Atlas sulfur analyzer is shown schematically in Figure 1. A sulfur-containing sample is fed continuously into the ceramic tube of the pyrolyzer where a t 1250-1300 "C in the presence of hydrogen the analyte is thermally cracked and sulfur converted to hydrogen sulfide. The exit gases are 0 1962 American Chemical Society
