included in this study. The optimum size of polymer sample was 3.0 g; however, some runs were made with as little as 200 mg. Biphenyl (in some instances, benzophenone) was used as an internal standard for quantitative analysis. Figure 1 is a typical chromatogram. Two common polypropylene stabilizers, 4-(dodecyloxy)-2hydroxybenzophenone (DOBP) and 2,6-di-tert-butyl-p-cresol (BHT), were quantitatively determined by this technique. The results, calculated from peak areas, from four separate runs are summarized in Table I. The precision for the BHT determination was good, but the quantity found (0.02 %) was less than the amount added (0.05%). BHT is very volatile, and some loss probably occurred during compounding. DOBP determinations ranged from 0.20 to 0.3.5z for a sample that originally contained 0.30%. These data reflect the poor resolution between DOBP and some other components that appear as shoulders on the DOBP peak (Figure 1). To demonstrate the scope of the method, a number of common polypropylene additives were determined by GLC (Table 11). Most of these additives were eluted without
Spectrophoto
decomposition in less than 1 hr; this group included 1,3,5trimethyl-2,4,6-tris(3,5-di-~ert-butyl-4-hydroxybenzyl)benzene, mol wt 774. However, pentaerythritol tetrakis(3,5-di-tertbutyl-4-hydroxyhydrocinnamate) mol wt 1058, although stable, was not eluted from the column within 1hr. A few compounds decomposed, as evidenced by peaks of low molecular weight fragments. For some compounds, such as dilauryl 3,3'-thiodipropionate, thermal instability could be predicted from the structure of the additive; however, such a prediction is not always possible. The marked difference between the unstable 2,2 '-thiobis[6-tert-butyl-p-cresol] and its stable isomer, 4,4'thiobis[6-tert-butyl-m-cresol], was unexpected. For additives other than those listed, the thermal stability must be determined by experiment. For a number of commonly used polypropylene stabilizers, this GLC method provides a rapid, quantitative determination and requires only a small sample of polymer. RECEIVED for review July 17, 1969. Accepted October 2,1969.
ination of Glyoxal iazolinone Hydraz
Fred W. Neumann Analytical Laboratory o j The Midland Dizision, The Dow Chemical Company, Midland, Mich.
IT IS OFTEN important to determine glyoxal when it occurs in biological systems, as a hydrolysis product, or as an oxidation intermediate in systems containing only two adjacent carbon atoms such as triethanolamine. Glyoxal can conveniently be determined at the parts-per-million level in aqueous and organic media by formation of the diazine of 3-methyl-2benzothiazolinone hydrazone (MBTH). Glycolaldehyde interferes in the semiquantitative method of Blumenfeld, Paz, Gallop, and Seifter (1). To avoid this interference the method was modified, and a quantitative procedure is described for the determination of glyoxal in the presence of glycolaldehyde and triethanolamine. The molar absorptivity of the pure diazine is determined for the first time. Formation of the diazine in situ is shown to be quantitative. Differences in previously recorded, qualitative spectral characteristics are shown to be the result of varying amounts of water in the solvents. When triethanolamine is present, it must be converted to the amine sulfate to attain the desired spectral Characteristics of the diazine. EXPERIMENTAL
Apparatus. Cary Model 14 spectrophotometer and 1-cm cells were used. Reagents. MBTH, 0.4%. Dissolve 0.40 f 0.01 g of 3-methyl-2-benzo-thiazolinone hydrazone hydrochloride monohydrate (Aldrich Chemical Company) in 100 ml of 80% acetic acid, and filter. The reagent should be prepared fresh daily, or else stored in a refrigerator to keep it from discoloring. GLYOXAL, 50 ppm standard solution. Dilute 20 ml of 40% glyoxal (Matheson, Coleman and Bell 7204) to 250 ml using distilled water and determine the exact concentration (1) 0. 0. Blumenfeld, M. A. Paz, P. M. Gallop, and S . Seifter, J. Biol. Chem., 238, 3835 (1963).
using the caustic-titration method of Salomaa (2). The calculated amount of this solution is then diluted to l liter using 80% acetic acid. GLYCOLALDEHYDE (HOCH2CHO) was obtained from Aldrich Chemical Company. A purified sample of diazine was prepared using 40% glyoxal and 3-methyl-2-benzothiazolinone hydrazone hydrochloride in water at room temperature. The crude product was recrystallized using dimethyl sulfoxide, washed with methanol, and dried at 105 "C under 5 mm Hg pressure. Yellow needles were obtained, mp 296-299 OC; the reported value is 296-297 "C (3). Anal. Calculated for ClsHlaN$32: C, 56.8; H, 4.24; N, 22.1; S, 16.9. Found: C, 56.0; H, 4.17; N, 22.2; S, 17.0. Differential scanning calorimetric analysis ( 4 ) indicated 99.5 mole per cent purity. The molar heat of fusion was calculated to be 13,094 calories. Calibration. Appropriate dilutions in 80% acetic acid were made using water, acetic acid, and a stock solution of the pure diazine (6.09 mg/100 ml acetic acid). The net absorbances at 407 mp were measured using 80% acetic acid as reference. The net absorbance was linear in the range of 0-1200 pg diazine per 100 ml; the molar absorptivity is 46.4 x 103. Procedure (For Triethanolamine). Weigh 1.OOO-1.030 & 0.001 g sample into a 50-ml volumetric flask and add the calculated amount of 1N sulfuric acid to neutralize the base present; 6.70 ml is required per gram of triethanolamine. Then pipet 2.50 ml of the 0.4% MBTH reagent into the flask and dilute the mixture to volume using 80% acetic acid. Shake well and allow to stand at room temperature for at least 2.5 hours. Concurrently a reference solution is pre(2) P. Salomaa, Acta Chem. Scarad., 10, 306 (1956). (3) G. Henseke, G. Hanisch, and H. Fischer, Ann. Chem., 643.3, 161 (1961). (4) C. Plato and A. R. Glasgow, Jr., ANAL.CHEM., $1, 330 (1969).
ANALYTICAL CHEMISTRY, VOL. 41, NO. 14, DECEMBER 1969
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20.19
Table I. Determination of Glyoxal in Known Mixtures Color development Glyoxal, pg Sample Time Temp, "G Added5 Found Room Water 2 . 7 hr 50 48* 3 min 100 50 490 60 3 min 50 50 f I d Room Triethanol4 hr 0 1.4,0.? Room aminea 4 hr 25 26 Room 4 hr 50 52 Room 4 hr 90 94 Room 2 . 5 hr 25 26 a Added the calculated aliquot of the standard 50-ppm glyoxal solution to sample, totaling 1 gram. * Found 51 pg after heating on steam bath an additional 3 hours. c Mixture heated with minimum acetic acid, then cooled and diluted to volume using 80% acetic acid. d Calculated from absorption readings taken at intervals from 25 minutes to 2.5 days at room temperature after heating period. e Commercial sample redistilled under reduced pressure. Assayed 99.3 & 0.4% by nonaqueous perchloric acid titration after acetylation. pared by diluting 6.7 ml of water and 2.50 ml of 0.4% MBTH reagent to 50.0 ml using 80% acetic acid. The net absorbance at 407 mp is measured using this reference solution. The glyoxal content is calculated using the value, 125.0 pg glyoxal per 100 ml per 1.0 absorbance unit. RESULTS AND DISCUSSION
The results in Table I show the method is applicable to the determination of glyoxal in commercial triethanolamine and in water samples in the range of 1 to 90 ppm. Glyoxal reacts quantitatively with MBTH in acetic acid as shown in Equation 1 to form a yellow dye with an absorption maximum at 407 mp.
+ CHO I
\C===="H, /
---b
CHO
1
i Unsubstituted monoaldehydes and monoketones will not interfere because they form colorless azines with the MBTH reagent ( I ) ; excessivelylarge amounts could interfere by consuming the reagent. Diacetyl, CH3@OCOCH3,and methyl glyoxal, CH3CQCH0, are known to give yellow derivatives of similar characteristics (3) and are thus expected to interfere if present. The presence of an oxidizing agent might interfere by converting the yellow dye to a blue dye (5). Mild oxidative conditions were not deleterious in our work. The recommended procedure is adapted from a semiquantitative procedure of Blumenfeld, Paz, Gallop, and Seifter ( I ) for determination of glyoxal in oxidized protein glycosides. Blumenfeld et al. developed the diazine by heating at 80 "C for I5 to 40 minutes in 50% acetic acid after adding MBTH. It has now been found that if the dye is developed by heating at (5) E. Sawicki, T. Hauser, T. Stanley, and W. Elbert, ANAL. CKEM., 33, 93 (1961). 2078
e
90 "C for 15 minutes or by allowing it to stand several days at room temperature, the presence of 100 ppm of glycolaldehyde in samples produced additional diazine, giving results high by 9 to 18 ppm. The presence of glycolaldehyde did not interfere when the absorbance was measured after reacting 2.5 hours at room temperature as recommended. Blumenfeld and coworkers found that longer heating periods were sometimes required to obtain maximum values, and concluded that glyoxal was slowly liberated from its glycosidic attachment. Our findings suggest that the presence of glycolaldehyde or its glycoside could also account for the apparent increased glyoxal content upon prolonged heating. Blumenfeld and coworkers utilized 50% acetic acid. The modified procedure utilizes 80% acetic acid which is considered a better solvent for organic samples. Admixture of triethanolamine containing 400 ppm of glyoxal and aqueous 0.4% MBTH reagent produced a white precipitate within 1 minute; later dilution with acetic acid showed very little yellow color as compared to a companion test containing 80% acetic acid at the start. Thus the reaction in the absence of acetic acid is considerably slower because of formation of an insoluble intermediate. The spectral characteristics were carefully studied because the literature references at first appeared contradictory. Henseke and coworkers (3) reported a major absorption doublet with peaks at 398 and 417 mp using 9 6 x ethanol as solvent. Blumenfeld and coworkers (1)reported a single peak at 405 mp in 50 % acetic acid. The spectral characteristics were dependent on the amount of water in the solvents and upon additives, and the differences were not due to geometric isomers associated with multiple conjugation of the azine group as suggested by the work of Dale and Zechmeister (6) in the cinnamalazine series. Using 50 to 94% acetic acid, the molar absorptivity is fairly constant (45.9 0.5) X los, with a single maximum changing from 406 to 410 mp as the acetic strength is increased. Using 94 to 100% acetic, the molar absorptivity increases up to 21 i 1% with decreasing water content; at concentrations greater than 97% acetic acid, a doublet is observed at 397 and 414 mp. The 397-mp peak is first discernible in 97% acetic. The described doublet in 96% ethanol (3) was also obtained using absolute ethanol as solvent; the doublet was still pronounced, but not as distinct in 80% ethanol. A singlet peak at 410 mp was observed using both 33 and 50% ethanol. The inclusion of 2 % triethanolamine as the €ree base when using 80% acetic acid produces a flattening of the peak and significant increase in absorbance. Prior neutralization to form the amine sulfate produced the described singlet. A kinetic study showed that diazine formation is complete in 2.5 hours at room temperature, and that the absorbance then did not significantly change after two to three days storage when glycolaldehyde was not present. The anticipated spectral characteristics were obtained when commercial triethanolamine samples and an aerated ethylene glycol sample were analyzed using the modified procedure. The amount of glyoxal found in three triethanolamine samples ranged from 0.9 to 7.4 ppm and 645 ppm in the glycol sample. Dye colors could be further developed by heating at 90 "C, and the resulting calculated values increased to 2.8-18 and 1160 ppm, respectively. The increases are attributed to the presence of glycolaldehyde. RECEIVED for review August 25, 1969. Accepted October 2, 1969. (6) J. Dale and L. Zechnieister, J . Amer. Chem. Soc., 75, 2319 (1953).
ANALYTICAL CHEMISTRY, VOL. 41, NO. 14, DECEMBER 1969
