Determination of Mixtures of Hydrazine and Monomethylhyd razine by Reaction with Salicylaldehyde NICHOLAS M. SERENCHA, J. GORDON HANNA, and EDWARD J. KUCHAR Olin Mathieson Chemical Corp., 275 Winchester Ave., New Haven 4, Conn.
)Mixtures of hydrazine and monomethylhydrazine are resolved in glacial acetic acid by reaction with salicylaldehyde in the presence of excess perchloric acid. In this system any hydrazone formed by the reaction of salicylaldehyde and monomethylhydrazine is hydrolyzed back to the original substances. However, the excess perchloric acid does not inhibit the forrnation of the azine. The perchloric acid neutralized by the sample is a direct measure of monornethylhydrazine. The hydrazine content is then determined by difference from a total base titration.
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involving samples containing monomethylhydrazine (MMH) and hydrazine (NzH4) prompted an analytical investigation to develop a method of analysis to quantitatively differentiate these components. At. the inception of our investigation the method of Clark and Smith (3) was the only one available for the analysis of such mixtures. Their method, which employs oxidation with Chloramine-T and sodium hypochlorite to differentiate M M H and K2H4, was tried initially on the experimental system; however, high, erratic results A recent were usually obtained. publication by Sutton (6), which utilizes gas chromatographic and wet analysis for the analysis of this mixture was not tested on the experimental system, Malone (4) analyzed mixtures of N2H4 and 1,l-dimethylhydrazine (UDMH) based on the reactions of these compounds with salicylaldehyde in acetic acid to form what was termed the neutral azine and the basic hydrazone, respectively. The basicity of the system was then determined and calculated as a measure of the U D M H and the NsH4 content' was obtained by difference from a total base titrat,ion. Malone attempted this method on the KZH4-Mh1H system and concluded that it was not successful because the reaction producing the hydrazone was incomplete. The evidence presented in the present study shows that the species XPERIMEXTAL
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ANALYTICAL CHEMISTRY
Figure 1 . Hydrazine reacted with salicylaldehyde and excess perchloric acid titrated with 0.1N sodium acetate
actually titrated are the free ,MMH and the free U D M H and that low recoveries are the result of incomplete hydrolysis of any hydrazone present during the titration. When an excess of perchloric acid is included in the salicylaldehyde reaction mixture, hydrolysis is complete and M M H can be determined. EXPERIMENTAL
Reagents. Perchloric acid, 0.1N in glacial acetic acid. Acetic anhydride is omitted in the preparation to avoid a n y acetylation of the hydrazines. T h e samples of hydrazine and monomethylhydrazine used in t h e study were obtained from the Chemicals Division of the Olin Mathieson Chemical Corp. and were employed without purification. Preparation of Sample. About 0.4 gram of N2H4 and/or -MMH is weighed to the nearest 0.1 mg. and added to approximately 35 ml. of chilled acetic acid in a 50-ml. volumetric flask. After equilibration to room temperature, the samples are diluted to 50 ml. with acetic acid and mixed thoroughly. Determination of Total Alkalinity. TITRATION .I. A 5-ml. aliquot is dissolved in 50 ml. of acetic acid, to which 25 ml. of 0.1N perchloric acid in acetic acid is added. The
solution is then titrated potentiometrically with 0.1N sodium acetate in acetic acid. The amount of perchloric acid consumed in Titration A b y the sample is a measure of t h e total hydrazine content (substituted and unsubstituted). Determination of MonomethylB. T o a sechydrazine. TITRATION ond 5-ml. aliquot dissolved in 50 ml. of acetic acid, 25 ml. of 0 . l N perchloric acid is added, followed by 2 ml. of salicylaldehyde. The solution is stirred on a magnetic stirrer for 4 t o 5 minutes, and is titrated as above with 0.1N sodium acetate. The amount of perchloric acid neutralized by the sample is a direct measure of monomethylhydrazine. Blanks are run in both titrations, omitting only the sample. CALCULATIONS
yo M M H
=
(meq. NaOAc Blank Titration B meq. NaOAc Titration B) 46.075 sample wt.
% NZH4
=
(meq. NaOAc Blank Titration A meq. NaOAc Titration A) (meq. XaOAc Blank Titration B mea. XaOAc Titration B) 32.05 sample wt.
The determinations should be made immediately on dilution of the sample with acetic acid to minimize hydrazide formation. A definite decrease in basic equivalents was noted for diluted samples which were permitted to stand longer than two hours a t room temperature. DISCUSSION AND RESULTS
Effect of Concentrations of Hydrazine and Monomethylhydrazine. I n the analysis of mixtures of NzH4and M M H with the above procedure, two distinct inflections are observed when the hydrazine concentration is greater than 50%; the first inflection is the excess perchloric acid, and the second inflection is the azine perchlorate salt (Figure 1). However, with less than 50Oj, hydrazine in the mixture, only one distinct inflection is observed, but a second constituent is very evident from the shape of the titration curve. Visual end point detection was not used in this system; however, the system should be amenable to a n indicator such as crystal violet. The procedure was tested by analyzing synthetic mixtures of NZH, and S I M H from O-lOOyGof each component. The data are tabulated in Table I and are corrected for x2H4 found in the M M H sample. Mechanism of the Reaction. The azine and hydrazone reactions are reversible and dependent on hydrogen ion concentration ( I , $ , 6). I n glacial acetic acid in the presence of perchloric acid, the equilibrium is displaced completely in the direction of azine formation. However, the reaction of M M H and the formation of the hydrazone is not complete in the same
medium. This was established by the following experiments : The salicylaldazine and the salicylaldehyde hydrazones of M M H and U D M H were prepared in benzene and analyzed quantitatively by nonaqueous titrimetry in pyridine. U D M H was included in this study for purposes of comparison. The azine assayed 101% and the hydrazones 100%. The infrared spectra of these materials were consistent with that expected of the azine and the hydrazones. These materials were then dissolved in acetic acid and titrated with 0.1N perchloric acid in acetic acid. The azine titrated as a very weak base and was quantitatively recovered, whereas the hydrazones titrated as relatively strong bases, of which the U D M H hydrazone was quantitatively recovered while the M M H was only 60-70Oj, recovered. The potentiometric plots obtained in the titrations of the M M H and UDRiIH hydrazones are almost superimposable on the plots of M M H and UDMH. However, the basicity of these hydrazones should be weaker than MMH and U D M H becouse of the decrease in electron density of the nitrogen atoms. Therefore, it was concluded that perchloric acid shifted the equilibrium such that the M M H and UDMH were actually titrated and not the weakly basic hydrazones. The low recovery of M M H could be attributed to incomplete acid hydrolysis of the hydrazone. It was established that the azine is stable, the M M H hydrazone is somewhat stable, while the C D M H hydrazone is entirely unstable in acetic acidperchloric acid media. Additional data confirming the above conclusions were obtained by agitating the azine and hydrazones in excess aqueous hydrochloric acid. The solu-
Figure 2. Ultraviolet spectrum of monomethylhydrazone of salicylaldehyde before and after addition of perchloric acid left. Before addition of perchloric acid Right. After addition o f perchloric acid
Table 1. Analysis of Hydrazine-Monomethylhydrazine Mixtures
yc MMH Present Found 100.0 89.1 79 2 73 4 65 1 49 6 37 8 29.3 19.3 13 0 0
100.2 90.4 80 5 73 0 66 3 50 9 38 9 28.4 19.2 13 1 0
% NZH4 Present 0 10.9 20 8 26 6 34 9 50 4 62 2 70.7 80.7 87 1 100 0
Found 0.9 11.1
21 1 26 3 35 0 49 6 62 0 69.5 82 2 87 5 99 3
tion was extracted with benzene to remove any liberated salicylaldehyde, free azine, and hydrazones. The aqueous phase was then evaporated to a residue which was identified by infrared as the hydrochloride salts of M M H and U D M H in the case of the hydrazones, whereas no hydrazine hydrochloride was detected in the azine esperimentLe., the azine was recovered as such in the benzene layer. More definite proof was obtained on this system by observing the ultraviolet spectral changes of the azine and the hydrazones in acetic acid and in the presence of excess perchloric acid. Figure 2 illustrates the ultraviolet spectrum of the M M H hydrazone before addition of excess perchloric acid, and immediately after the addition of excess perchloric acid. About 94% of the hydrazone has hydrolyzed to M M H and salicylaldehyde. The spectrum after addition of excess perchloric acid is practically identical to that of free salicylaldehyde in acetic acid, with the exception of the small amount of material a t approsimately 288 mp which could be attributed to N2Hr present in the original h l M H since this absorption is still evident after 70 minutes, and it was established that the azine is stable for this length of time in this system. U D M H was also tested as above, and it was established that the U D M H hydrazone is also unstable to perchloric acid in acetic acid-i.e., the equilibrium is shifted completely in favor of free U D M H and free salicylaldehyde. The ultraviolet spectrum of the azine before addition of perchloric acid and immediately after the addition of perchloric acid shows a shift in the spectrum, but no free aldehyde is present immediately as shown by absence of the salicylaldehyde peak at 256 mp. After 24 hours in the presence of excess perchloric acid the azine begins to hydrolyze as indicated by the formation of the salicylaldehyde peak a t 256 mp. After 24 hours, 80% of the azine is hydrolyzed back to hydrazone and salicylaldehyde. VOL. 37, N O . 9, AUGUST 1965
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Therefore, the conclusion arrived a t by Malone-i.e., that the UDMH hydrazone of salicylaldehyde is as basic as UDMH-is incorrect. Under the conditions employed by Malone, the U D M H hydrazone is hydrolyzed rapidly to U D M H and salicylaldehyde upon addition of perchloric acid, whereas,the azine is stable and therefore not titrated as a moderate base in glacial acetic acid.
ACKNOWLEDGMENT
The authors gratefully thank Irene Cowern, Warren Harple, and George Hurt who performed the infrared and ultraviolet determinations.
(2) Clark, C. C., “Hydrazine,” Mathieson Chem. Corp. Baltimore, Md. (1953). (3) Clark, J. D., Smith, J. R., ANAL. CHEM.33, 1186 (1961). (4) Malone, H. E., Ibid., p. 575. (5) Sidgwick, N. V., “Organic Chemistry of Nitrogen,” p. 393, Clarendon Press, Oxford, 1949. (6) Sutton, N. V., Microchem. J . 8, 23 (1964).
LITERATURE CITED
( 1 ) Ardagh, E. G. R., Williams, J. G., J . Am. Chem. SOC.47, 2976 (1925).
RECEIVEDfor review February 17, 1965. Accepted May 28, 1965.
Separation of Protium and Deuterium Forms of Carbohydrates by Gas Chromatography RONALD BENTLEY, NRIPENDRA C. SAHA, and CHARLES C. SWEELEY Department of Biochemistry and Nutrition, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pa.
b Gas liquid chromatography on 15to 25-meter, narrow-bore packed columns of SE-30 and other liquid phases brought about the complete separation of trimethylsilyl derivatives of @glucose from 0-glucose-d,. Partial separations were achieved with the Q anomers of glucose and glucosed;, and with the a and @ anomers of other pairs of protium and deuterium sugars. Structural factors appear to play a significant role in these separations. It is calculated that columns with 200,000 theoretical plates would give complete resolution of all the protium-deuterium pairs studied (ribose, xylose, fucose, rhamnose, rnannose, and glucose).
T
possibility of fractionation in compounds containing isotopes of hydrogen has received considerable attention, particularly with the advent of high resolution chromatographic methods. (The term “fractionation” rather than “isotope effect” is used to describe the separations discussed here since the latter term is usually reserved for chemical reactions). One of the earliest studies of such a fractionation in a gas liquid partition chromatographic system was that by Rilzbach and Riesz in 1957 (20),in which cyclohexane and its completely deuterated form (CCDl2)were partially separated. More recently, Lee and Rowland (8) have demonstrated a partial resolution of monotritiated butene on a 160-foot column packed with silver nitrate-ethylene glycol. Root, Lee, and Rowland (12) have described an effective recycling technique for the separation of butane from deuterated butane, and methane from deuterated methane. Van Hook and Kelly (17) separated deuterated ethanes by gas chromatography, using HE
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ANALYTICAL CHEMISTRY
methylcyclopentane as the liquid phase; subambient operating temperatures in the range 273’ to 158’ K. were employed. Other chromatographic techniques have also been shown to lead to fractionation. For example, Cant and Yang (4)have partially separated all of the tritium-substituted methanes and deuterium-substituted methanes by gassolid adsorption chromatography on a 50-foot column packed with charcoal. h great many observations have been reported on the separation of molecular hydrogen, deuterium, and tritium from each other and from mixed forms such as H D and D T [for a review of this work, see Akhtar and Smith (I)]. Liquid-solid chromatography has been used by Klein, Simborg, and Szczepanik ( 7 ) to achieve some isotope fractionation of (214- and H3-labeled cholesterol acetates on silica gel columns. In this case, a measurable effect was noted with one tritium atom in the presence of 47 hydrogen atoms, in a compound of molecular weight 430. Similarly, Sgoutas and Kummerom (14) have shown small differences in C14- and H3labeled fatty acids on chromatography with silica gel-silver nitrate columns. Piez and Eagle ( 2 1 ) observed a slight fractionation on ion exchange chromatography of C14-labeled amino acids, and obtained evidence that the effect of the C14 atom on chromatographic behavior a a s dependent on its position in the molecule, rather than solely on the basis of a difference in mass. Similarly, Marshall and Cook ( I O ) noted that small differences in countercurrent distribution of arabinose-l-CI4 and other labeled pentoses were not related to differences in mass only. These effects have generally been demonstrated in gas chromatography
with compounds of relatively low molecular weight, in which the isotope-labeled species differed in mass from the protium form by from 5 to 10%. The only exception has been the recent report by Kirschner and Lipsett (6) of a slight separation of H3-and C14-labeled steroids on short packed columns. If complete separations were possible with isotope-containing compounds of higher molecular weight, chromatographic techniques might be used in the biomedical field for direct determinations of isotope abundance following metabolic tracer A direct chromatoexperiments. graphic procedure would obviate the necessity of converting compounds to a form suitable for isotope analysis by classical methods of mass spectrometry, and would be less complex, instrumentally, than recently described techniques for continuous mass spectrometric analysis of gas chromatographic effluent streams (13,18). Deuterated hexoses, in which all of the carbon-bound hydrogen is replaced by deuterium, are now commercially available. Since we had developed a rapid method for the preparation of 0-trimethylsilyl (TMS) derivatives of polyhydroxy compounds, and had studied the gas chromatographic behavior of many of these compounds (3, 16, 19), we undertook an investigation of the chromatographic separation of deuterated hexoses (and other sugars) from the protium forms. For a compound such as glucose, with seven carbon-bound deuterium atoms, the mass difference for the TMS derivative is 7 in 547, or 1.3%. This paper reports the complete separation of glucose and glucose-&, and partial separations with other carbohydrates, using gas liquid chromatographic columns with efficiencies of from 40,000 to 60,000 plates.
