V O L U M E 24, NO. 12, D E C E M B E R 1 9 5 2 removed from each sample under reduced pressure and the melting point of the resulting solid material determined. Consideration of the melting points of consecutive samples indicated that certain of these small samples could be recombined i n many cases and further purified by rechromatographing or by fractional crystallization techniques. The purified derivatives obtained by such methods were identified by melting points and mixed melting points with knovn derivatives and also by carbon-hydrogen analyses in certain cases. I n the fractions which contained more than one ester the relative amounts of each acid derivative were estimated from the quantity of each derivative obtained. An exact determination of the relative quantity of each derivative could not be made, since the p-phenylphenacyl derivatives of the high molecular weight acids in many of the fractions could not be separated completely on the chromatographic column or by fractional crystallization techniques. These data are summarized in Table V.
Identification of Free Acids. The free acids of the dried fusel oil were removed by passing the fusel oil slowly through a column of Amberlite IRA 400 resin n-hich was in the hydroxide form and which had been washed rrith 50% ethyl alcohol-water solution in order that the fusel oil would wet the resin. The higher alcohols were washed out of the column with successive portions of 50% ethyl alcohol-water solution and water, after which the anions were displaced by a wash with 10% sodium hydroxide solution. The excess sodium hydroxide and the sodium salts of the acids were then run through a column of Amberlite IR 120 which was in the hydrogen form. The effluent from the cation exchanger was neutralized with standardized sodium hydroxide solution. the excess water was evaporated on a steam bath, and the p-phenylphenacyl derivatives were prepared. The mixed derivatives were chromatographed by the method previously mentioned. Five blue fluoreficent zones were separated, two of which contained fairly large amounts of material. The material from the upper of these two zones rerrystallized from ethyl alcohol as
1949 colorless plates, melting point 108.8-110.0", mixed melting point 110.0'-lll.Oo with p-phenylphenacyl acetate. The lower large zone gave white plates on recrystallization from ethyl alcohol, melting point 79.7-80.8", mixed melting point 81.0-82.0' with p-phenylphenacyl-n-butyrate. ACKNOWLEDGMENT
The authors wish to acknowledge the financial assistance of the Wine Advisory Board in support of this research. They also wish to thank James F. Guymon for furnishing the fusel oil sample, and Amos Newton for making the mass spectrometric analyses. LITERATURE CITED
Castor, J. G. B., and Guymon, J. F., Science, 115, 147 (1952). Claudon, E., and Morin, E.-Ch., Compt. rend., 104, 1187 (1887). Dupont, Georges, and Dulou, Raymond, Ibid., 200, 1860 (1935). Ehrlich, F., Ber., 40, 1027 (1907). Horsley, L. H., ANAL.CHEM.,19, 531 (1947). Joslyn, hl. A., and ilmerine, M. A., Univ. Calif. Agr. Expt. Sta., Bull. 652 (1941). Kirchner, J. G., Prater, A. K., and Haagen-Smit. -4.J., IND. ENG.CHEY.,ASAL. ED.,18, 31 (1946). Morin, E.-Ch., Compt. rend., 105, 1019 (1887). Neubauer, O., and Fromherz, K., 2. p h ~ ~ i o Chem., l. 70, 326 (1911).
Ordonneau, Ch., Compt. r e n d , 102, 217 (1886). Shriner, R. L., and Fuson, R. C., "Identification of Organic Compounds," 3rd ed., p. 133,New York, John Wiley & Sons, 1948. Ibid:,-p. 157.
Thorne, R. S. W., J . Inst. Brewing, 55 ( n s . 461, 201 (1949). Ubeda, F. B., Anales fis. quina., 37, 356 (1941). Valaer, Peter, I n d . Eng. Chem., 31, 339 (1939). White, J. IT., Jr., and Dryden, E. C., ANAL. CHEM.,20, 853 (1948).
RECEIVED for review July 7 , 1952. Accepted September 22, 1952.
Analysis of Pyrazine Methods for Assay and Impurities WILLIAM SEAMAS, J. T. WOODS, AND WLADIMIR LEIB\IISN Calco Chemical Division, American Cyanamid Co., Bound Brook. V. J . As a part of a program of work on sulfapyrazine, a method was needed for determining pyrazine. A method is proposed whereby pyrazine is precipitated as a mercuric sulfate complex. The excess standard mercuric sulfate is titrated with thiocyanate. Piperazine, diethylenetriamine, ethylenediamine, and ethanolamine do not interfere. A small correction is applied for the solubility of the complex. The pyrazine values have a standard deviation of 3 ~ 0 . 3 %(absolute) and seem t o suffer no systematic error. Some indication of the amount of the above-mentioned basic impurities, and of the presence of unidentified impurities, is obtained by a potentiometric titration w-ith standard alkali after addition of an excess of standard acid.
I
K C O S X I X T I O S with work on sulfapyrazine, a method of analysis was needed \Thich would be able t o differentiate pyrazine from basic impurities that were likely to be associated nith it. KOsuch methods were found in the literature, but Stoehr ( I , 4 ) had reported that pyrazine formed an addition compound with mercuric chloride n-hich n-as insoluble in water and dilute acid. Utilization of this reaction as the basis of a convenient titrimetric method of assay of pyrazine would require titrating the excess mercuric chloride left after carrying out the reaction. Because chlorides interfere with the commonly used titration with thio-
cyanate ( 2 ) , mercuric sulfate \\-as used rather than mewuric chloride. A potentiometric titration gives an indication of the type and amount of the basic impurities, mainly piperazine, ethylenediamine, diethylenetriamine, and ethanolamine, which may be present as a result of the particular synthesis which was used. It cannot be interpreted in terms of any specific impurity, but does, nevertheless, indicate to a small degree which of the expected bases may be present. Water may be determined by the Karl Fischer reagent in the usual manner.
ANALYTICAL CHEMISTRY
1950 ASSAY METHOD
Reagents. Mercuric SulCate Solution. A solution of 30 grams of reagent-grade mercuric sulfate in 850 ml. of water and 150 ml. of concentrated sulfuric acid is filtered through glass wool and standardized against 0.1 N ammonium thiocyanate, using ferric ammonium sulfate as the indicator. Ammonium Thiocyanate Solution, 0.1 h'. The ammonium thiocyanate is standardized against a standard silver nitrate solution. Ferric Ammonium Sulfate. Ten milliliters of concentrated nitric acid are added to 125 grams of reagent-grade ammonium iron alum [Fer(S04)a. (SH4)2S04.24HzO] dissolved in 100 ml. of water. Procedure. The samples which were analyzed included both liquids and solids (pyrazine is a solid melting a t about 53" C.). The solid samples were melted in an oven a t 60" to 70' to ensure uniformity of sampling. A q-eighed 4- to 7-gram sample of the melt was dissolved in about 300 ml. of water and diluted to 1 liter in a volumetric flask. Fifty milliliters of standard mercuric sulfate solution was added to a 50-ml. aliquot dropwise from a buret, with mechanical stirring, over a period of 5 to 10 minutes. The reaction mixture was allowed to stand overnight a t room temperature. (If somewhat poorer precision is acceptable, the overnight digestion is not necessary.) The reaction mixture was filtered with suction and the precipitate of the mercury complex was washed with about 100 ml. of water. The filtrate was titrated with 0.1 N ammonium thiocyanate, using 4 ml. of the ferric ammonium sulfate solution as the indicator. The pyrazine content of the sample was calculated from the following equations:
+ HgSOa +HgSOa . C4H4S2 HgSOi + 2NH4CNS +Hg(CKS)z + (NH4)ZSOa CiHaNi
(1)
(2)
A solubility correction of 0.50 ml. of 0.1 N thiocyanate was added to compensate for the solubility of the pyrazine-mercuric sulfate complex. This correction is discussed below.
Table I.
Comparison of Rlercury Contents of Precipitates Obtained under Various Conditions
Conditione of Precipitation Dropwise addition of reagent Dropwise addition of reagent Dropwise addition of reagent Dropwise addition of reagent Rapid addition of reagent Rapid addition of reagent Rapid addition of reagent a Pyrazine present, 96.7%. b Theory, 53.2%.
Table 11.
Mercury in Pyrazine Mercury in Excess, Rleq. Foundo, % Precipitateb, % 5.3 96.4 52.9 3.0 96.8 53.6 1.4 96.5 53.0 0.4 97.0 52.5 4.0 100.8 52.6 2.5 98.9 52.5 0.5 98.1 52.6
Effect of Temperature of Precipitation
Conditions Recommended method
Av. Precipitated a t boiling point under reflux Precipitated hot in open beaker
3
.-I 4
P
a
48
56
Figure 1. Potentiometric Titration of Impurities ImDuritv . Piperazine Diethylenetriamine Ethylenediamine Ethanolamine
Curve
A E C
D
Weieht. - . G. 0 2450 0.1946 0.1402 0.2046
for the purity of the pyraaine is shown in column 4 of Table I. These data fail to disclose any evidence that the high values are caused by the precipitation of a complex containing a higher concentration of mercury. The higher values obtained by the rapid addition must therefore be due to the precipitation, because of local excesses of mercury, of mercury complexes of some of the impurities which are present in the sample. An attempt to eliminate this difficulty by precipitating hot in an open beaker, rather than a t room temperature, led to low values. Under reflux there was some suspicion of lowered values. Losses of pyrazine on heating might occur because of the existence of an azeotrope with water which boils a t 95.5" C. (uncorrected) ( 3 ) . A comparison of the values obtained under these conditions is given in Table 11. Solubility Correction. The solubility correction was determined by suspending 50.0 mg. of the complex and varying amounts of mercuric sulfate in the same volume of solution and under the same conditions of acidity as those existing during the assay. After occasional shaking and then standing overnight, the remaining precipitate was filtered off and washed with 100 ml. of water and the mercuric sulfate in the filtrate was determined by means of ammonium thiocyanate in the usual manner. The values obtained are shown in Table 111. From this it can
Pyrazine Found, % 97.0, 96.5, 96.8, 96.4, 96.7 96.2, 96.2 86.9, 80.6
Method of Precipitation. The effect of the amount and manner of addition of mercuric sulfate is shown in Table I. For a dropwise addition of mercuric sulfate the values obtained do not depend on the excess of mercuric sulfate, over the range 0.4 to 5.3 meq. If the reagent is added more rapidly, such as in a steady stream from a pipet, higher values are obtained. To attempt to determine whether these high values are due to coprecipitation of impurities or to the formation of complexes containing greater amounts of mercury, the mercury was determined in some of the precipitates by dissolving about 0.2 gram of the precipitate(previous1ydried a t l l O o C.)in 20 ml. of concentrated nitric acid, diluting ten times with water in order to obtain a solution about 1.5 S in acidity, and titrating with ammonium thiocyanate, using ferric ammonium sulfate as the indicator. .4 comparison of the mercury values for the precipitates and the values obtained
94 32 40 0.1 N NaOH, MI.
16
I
Table 111. Solubility of Pyrazine-Mercuric Sulfate Complex Excess HgSOd. as 0 . 1 hr N H L N S Solubility of Temperature MI.0.1 N Consumed b y HgSOi Complex, as A l l . C. NHICXS Complex, Ml. 0 . 1 N NH&NS Room 10.16 10.64, 10.64, 10.59 0.48, 0.48, 0.43 Room 39.56 40.10, 40.14, 40.14 0.54, 0.58, 0.58 Room 61.31 61.93, 61.98, 61.94 0.62, 0.67, 0.63 5 40.71 41.13, 41.22, 41.25 0.42, 0.51, 0.54 35.3 40.71 41.22, 41.23, 41.21 0.51, 0.52, 0.50 AY.
+
Table IV.
Effect of Aging of Precipitate
Digestion a t Room Temperature, Hours 0 1 2 16-20 a
hv 0.46 0.57 0.64 0.49 0.51 0 . 53
48 Pyrazine present, 98.6%.
Pyrazine Found, %" 99.0, 98.2 98.9, 99.8, 98.5 98.8, 99.5 98.3, 98.5, 98.7, 98.5, 98.6, 98.6, 98.3, 98.5 99.2, 98.8
Av. 98.6 99.1 99.2 98.5 99.0
V O L U M E 24, NO. 12, D E C E M B E R 1 9 5 2
1951
be seen that the solubility correction is reproducible and does not depend upon the temperature of precipitation. I t may possibly depend to some extent upon the excess of mercuric sulfate but the data involved do not indicate this with certainty. Precision and Accuracy. The precision of the method de-
Table Y. Effect of Impurities Pyrazine Founds Gram Pyraeine Found Gram ' Gram Pyraeine Present 0.3050 101.0 0.120 0.3022 100.1 Ethylenediamine 0.015 0.3030 100.3 0.030 0.3006 99.5 Diethylenetriamine 0.060 0.3018 99.9 0.120 0.3030 100.3 Et hanolamine 0.060 0.3014 99.6 100.6 0.120 0.3038 a 0.3020 gram of pyrazine present. Substance -4dded Piperazine
.4dded Gram' 0.060
loo
pends to some extent on the sampling and the time of digestion of the precipitate. For best conditions-that is, overnight digestion and using aliquots of the same volumetric solutionthe standard deviation of a single value from its mean is +0.3V0 (absolute). This value is calculated from data on four samples with four to eight replicates on each. If the solutions are filtered within a period of 2 hours, no systematic error is encountered. but the precision may be somewhat poorer. An indication of the effect of time of precipitation with aliquots of the same solution is shown in Table IV. In regard to accuracy, there is no reason to believe that the method suffers from any systematic errors. A correction is applied for solubility of the complex, and the impurities most likely to be present, mainly diethylenetriamine, ethylenediamine, ethanolamine, and piperazine, do not interfere when amounts even up to 20 to 40% are present. The values obtained for the determination of pyrazine in the presence of these impurities are shown in Table V. A sample of pure pyrazine was not available, but a value of 99.1% pyrazine was obtained for one sample which contained 0.0870 water and small amounts, probably tenths of 1%, of basic impurities. METHOD FOR IiMPURITIES
10
Basic substances that may be present in pyrazine because of the method of synthesis which was used may be determined by a potentiometric titration. The titration curves for the substances most likely to be present are shown in Figure 1. These curves have inflection points in the regions of pH 4 and 8. However, as there are a t least four likely impurities and only two inflection points, it is impossible to give an unequivocal answer in terms of any one impurity or even any one type of impurity.
8
4
P
Procedure. A weighed sample of 2 to 5 grams is dissolved in 50.0 ml. of 0.1 X hydrochloric acid and titrated potentiometrically with 0.1 A' sodium hydroxide, using a glass-calomel electrode system and a Beckman Model G pH meter. The shape of the curve obtained depends upon the amount and kind of the impurities present, but all of the samples exhibited two inflection points (Figure 2 ) , one in the region of pH 4 and the other in the region of pH 8. PIIilliliters of 0.1 Nsodium hydroxide to the inflection point a t pH 4 may be expressed as C and to pH 8 as D. As several impurities may be present, the curve cannot be interpreted in terms of any one amine, so that it is necessary to report the results in terms of milliequivalents of alkali per gram of sample,
t 38
Figure 2.
40
4P 44 46 0.1 N NaOH, MI.
48
SO
Titration of Impurities in Pyrazine Samples
Sample in 50 nil. of 0.1 S HC1 titrated with 0.1 N XaOH Curve Sample Weight, G. 8 1.76 A 1 2.18 B 2.53 5 C 2 2.35 D Blank E
E = milliequivalents of alkali consumed between pH 4 and 8 per gram of sample = - , - . LJ-c
Table TI. Sample 1
2 3
4
5 6
7 8
Pyraeine, yo 97.0, 96.5, 96.8, 96.4 Av. 96.7 98.6, 98.9 Av. 98.8 98.3, 98.5, 98.7, 98.5, 98.3, 98.5, 98.6, 98.6 Av. 98.5 99.4, 99.8, 98.9, 98.5 A v . 99.2 96.5, 96.0 Av. 96.3 91.1, 9 2 . 8 Av. 92.0 52.3, 52.6 Av. 52.5 94.5, 94.8, 94.8, 94.5 Av. 94.7
Summary of Results E
F
0.174,0.170 0.172 0.044
0.101,0.117 0.109 0.044
YOWater
Melting Range, O C. (Cor.)
0.23 0.19
49.5-52,2
0.21 0.20, 0.20 0.20
51.6-53.1
...
...
...
0.019
0.023
0.082 0.080
... 51.4-53.1
0.081
0.137, 0.146 0.142 0.276
0.030, 0.038 0.034 0.028
.
...
...
...
3.03
1.89
...
0.302
0.324
0.14, 0.15
(Liquid a t room temp.) 45.9-50.4
,,
0.15
10 X weight of sample in grams 50.0 - D F = 10 X weight of sample in grams
(3)
(4
G = total milliequivalents of alkali consumed per 50.0 - C gram of sample = 10 X weight of sample in grams (5) Discussion. G includes all the basic substances which may be present in the sample. E includes one equivalent of any di-acid base such as ethylenediamine and piperazine, and one equivalent of a tri-acid base such as diethylenetriamine. F includes strong mono-acid bases such as ethanolamine, one equivalent of piperazine and ethylenediamine, and two equivalents of diethglenetriamine. From Table VI, a summary of the analysis of several samples, and from Figure 2 it may be seen that most of the samples contain relatively small amounts of these basic substances in view of the equivalent weight of the rompounds concerned, Thus, samples 2 and 4 probably contain only a fern tenths of 1%of these bases, while samples 1,5, 6, and 8 contain considerably more, possibly I to 2y0. Sample 7 contains considerably more im-
1952
ANALYTICAL CHEMISTRY
purity than any of the other samples. It was a liquid IThile all the others were solids. I n addition, it would appear that some of these samples contain an impurity n-hich is not any of those considered, as all the latter compounds would give values for F which ~ o u l dbe either equal to or greater than E. It is probable that these unknown impurities are xeaker bases than those being considered. Determination of Water. Kater may be determined in the various pyrazine samples by the Karl Fischer reagent in the usual manner. A stable end point could be obtained. LITERATURE CITED
(1) Beilstein, F., “Handbuch der organischen Chemie,” 4te Auflage,
Bd. XXIII, p. 91, Beilin, Julius Springer, 1 9 3 6 ; -4nn Arbor Mich., Edwards Bros., 1944. Hillebrand, T. F., and Lundell, G. E. F., “Applied Inorganic Analysis,” p. 172, S e w York, John Wiley &- Sons, 1929. Pfann, H. F., J . Am. Chem. SOC.,66, 155 (1944). Stoehr, J . prakt. Chem., 51, 459 (1895). RECEIVED for review April 21, 1952. Accepted September 4 , 1952. Presented before the Division of dnalytical Chemistry a t the 121st Meeting of the AXERICAN CHEMICAL SOCIETY. Buffalo, K. Y., and before the Meetingin-Miniature of the North Jersey Section, AMERICAACHEMICAL SOCIETY, January 9, 19.50.
Paper Chromatographic Separation and Determination of Some: Water-Soluble Vitamins ‘
JAMES A. BROWN AND RlAX 41. MARSH Eli Lilly and Co., Indianapolis, Ind. With the large number of multiple vitamin preparations now being manufactured, the problem of adequate routine analytical control of these mixtures is becoming increasingly complex. As a potentially simpler general technique, paper chromatographic separation procedures for various vitamins were studied. It was found that four of the commonly encountered B complex vitamins-thiamine salts, riboflavin, nicotinamide, and pyridoxine hydrochloridewere readily separable on paper strips in a butanol-acetic acid-water system. By means of an automatic scanning device adapted for the Cary
E
XAMIKATIOS of the literature reveals the work of many on paper chromatography. It has become an accepted tool of the analytical chemist for the separation of closely related materials. K i t h particular reference to vitamins, Hais and Pec&kovB( 7 ) have used this procedure for separating riboflavin, Beran and Sicho ( 1 ) for thiamine, Kodicek and Leddi (9) and Huebner (8) for nicotinamide, and Wollish, Schmall, and Shafer (13)for nicotinic acid. I n this paper it is shoTYn how four synthetic vitamins of the B complex-thiamine salts (hydrochloride or monitrate), riboflavin, nicotinamide, and pyridovine hydrochloride-can be simultaneously separated, identified, and quantitatively estiniated by means of the paper chromatographic technique, the apparatus for automatically scanning paper strips with the Cary recording spectrophotometer described by Parke and Davis (IO), and the correlation of area measurements of the peaks on the resulting graphs. Somewhat similar methods for the quantitative evaluation of components separated on a paper strip have been described ( & - 6 , I I , I 2 ) . All of these make use of a relation between concentration and a measure of zone dimension-Le., maximum absorbancy, total absorbing area, or length. I n all cases, precision has been rather unsatisfactory and the discontinuous process of measurement very tedious The present method for plotting the absorbancy of the zones continuously in order to obtain accurate zone measurements is rapid and the precision obtainable is much greater than that outlined in other procedures. EXPERIMENTAL
The paper chromatographic portion of the work involved the development of chromatograms on 17/32 X 22.5 inch strips of Whatman S o . 1 filter paper cut parallel to the grain of the paper. Bscending technique vias used, the solvent being placed in the
recording spectrophotometer, curves were plotted of total ultraviolet absorption us. position on the strip. Quantitative results were obtained by measuring the areas of the discrete zones of absorption corresponding to each component and comparing them to those obtained from standard solutions. Preliminary studies indicate that the quantitative technique devised is sufficiently reproducible to be useful as a control method for certain types of pharmaceutical preparations. A single procedure may be utilized to analyze mixtures of many different and not necessarily related compounds.
bottom of 12 X 24 inch cylindrical glass jars having flat, closefitting glass lids. The jars were wrapped with opaque paper to prevent the destructive action of light. Jars made of nonactinic red glass have subsequently been obtained and are more convenient to use. The solvent system finally chosen was the upper phase obtained by shaking together 40 parts of reagent grade n-butyl alcohol, 5 parts of reagent grade glacial acetic acid, and 55 parts of distilled water (by volume). This solvent is similar to that of Hais and Pecitkovit ( 7 ) , but contains less acetic acid. The vitamins (purest commercial grade available)-individually and in mixtures-were dissolved in 50% (v./v.) acetic acid in water (heating, if necessary) and finally diluted so the resulting solution was 10% (v./v.) acetic acid. A 0.0175-ml. sample was applied 1.5 inches from the end of each strip. It was measured with a capillary pipet made from a common 0.09-ml. antibiotic pipet. The chamber of an antibiotic pipet was drawn out to a small volume and the outside dimension of the tip was reduced to about 1.5 mm. The chamber was filled by capillary action and discharged by the capillary action of the paper strip when the strip was touched to it. I t was calibrated with distilled water, using a semimicrobalance to weigh the water delivered, and Tvas found to discharge 0.0175 =k 0.0002 ml. The strips were dried in a 50’ C. oven after the sample wae applied, 10 minutes’ time in the oven being sufficient. They were then suspended in the jar with the end containing the sample dipping into the solvent in the bottom of the jar to a depth of approximately 0.25 inch. The lid was lubricated with stopcock grease and weighted to make a tight fit. The strips were developed overnight (I5 hours) a t room temperature and then dried in a 50” oven for 15 minutes. The principal scanning to provide measurable areas was performed at predetermined wave lengths; absorbancy us. a linear function of strip length was recorded as outlined by Parke and Davis ( I O ) . The instrument was set near its maximum sensitivity, the slit setting being about 2.75 mm. The slit width changed somewhat during the course of a run to compensate for voltage or light intensity changes. Figure 1 shon-s the graphs of a typical strip both before and after development.
