Simple Tests for Identification
OF
Sulfonamides
ALBERT B. SAMPLE Research Laboratories, Smith, Kline and French Laboratories, Philadelphia, Pa.
Simple tests depending on the formation of colored precipitates or solutions with cobalt and copper salts are adapted to the identification of sulfacetimide, sulfadiazine, sulfamerazine, sulfamethazine, sulfanilamide, sulfapyrazine, sulfapyridine, sulfaruxidine, and sulfathiazole. Other alkali-soluble sulfanilamide derivatives would b e expected to give similar color reactions with these reagents. Sulfaguanidine and other alkali-insoluble derivatives can b e identified b y chemical and physical properties which appear elsewhere in the literature.
T
ESTS for sulfonamides involving reaction of their sodium salts with copper sulfate have appeared in the literature (1,4, 5 ) , but, as presented, they do not afford positive differentiation. The purpose of this report is to present a standardized technique for these and supplementary tests, by means of which the most widely employed sulfonamides are easily identified. Lott and Bergeim (4) used this copper sulfate reaction in distinguishing between sulfapyridine and sulfathiazole and characterized the reaction products by means of elemental analysis. They also proposed the quantitative determination of these two drugs, as well as sulfanilamide, by ashing the copper sulfate reaction products and weighing the resulting cupric oxide. The United States Pharmacopoeia (5) and the Council on Pharmacy and Chemistry of the American Medical Association (1) make use of the reaction of copper sulfate with the sodium salts of sulfonamides in identifying the latter. Their descrip tions of colors for the reaction products, however, are a t times misleading and their procedures do not permit reasonably close duplication of results. PRINCIPLE OF M E T H O D
On the addition of a solution of copper sulfate or cobalt nitrate to solutions of the alkaline salts of sulfonamides, colored precipitates or solutions, which can be differentiated visually, are produced. Since most of the copper-sulfonamide precipitates go through series of color changes which a t some stages are similar for different sulfonamides, it is advisable to confirm results of these tests, either by timing approximately the periods of color changes of the copper-sulfonamide precipitates or by preforming the cobaltr sulfonamide tests for supplementary information. A further confirmatory test is necessary for sulfanilamide, since it is a t times impossible to distinguish between its copper precipitate and a precipitate of copper hydroxide. For this purpose, use is made of the fact that the copper-sulfanilamide precipitate when dissolved in sodium hydroxide and treated with potassium cyanide produces an amber solution, whereas a copper hydroxide precipitate when similarly treated produces a colorless solution. PROCEDURE
Five milliliters of 0.1 N (approximate) sodium hydroxide are measured into a 20 X 150 mm. test tube, and solid unknown material is added in small amounts until there is a definite excess and no more will dissolve. The solution is filtered throu h B wet No. 1 Whatman filter paper (7 cm.) and the filtrate cofected in a similar test tube If the filtrate is not clear, it is refiltered through a double thickness of wet filter paper or through a more retentive paper. Three drops of 15% cupric sulfate pentahydrate or 3 drops of 15% cobaltous nitrate hexahydrate are added to the filtrate. To identify the unknown sulfonamide, the precipitate or color formed is compared with the descriptions given in Tables I and 11.
DISCUSSION
Some of the tests described have been used in this laboratory for more than a year and a half, and t%e color descriptions given have proved adequate to make the tests adaptable for routine use on sulfonamide powders, tablets, and suspensions. Serial color changes are gradual, and time intervals given for such changes are only approximate. Color descriptions should be used only as a guide by individuals in establishing their own impressions of the colors. Colors of precipitates may vary slightly with variations in particle size, but, as a rule, such discrepancies are insignificant. If there is any doubt about the identification of an unknown by this procedure, the test should be repeated with known sulfonamides run simultaneously and under the same conditions. It would be expected that other alkali-soluble sulfanilamide derivatives would give similar specific reactions with the cobalt and copper reagents.
Table
1.
Cupric Ion-Sulfonamide Color Changes Total Time
Sulfonamide Sulfacetimide
Light Light Light Faint Light
Observed Changes in Order of Appearance yellow-green precipitate yellow-green opalescent solution yellow-green gelatinous precipitate greenish-blue greenish-amber sediment supernatant}
No change Yellow precipitate Yellow-green precipitate Green precipitate Green-brown precipitate Brown precipitate Purple-brown precipitate Brownish-purple precipitate Brownish-purple sediment Cloudy supernatant N o change Sulfamerazine Light yellow-green precipitate Deep green, clear solution Gray-green, turbid solution Gray precipitate Gray-brown precipitate Blackish-brown precipitate Gray-brown sediment Purple-brown supernatant} N o change Sulfamethazine Yellow-green precipitate Dark green, turbid solution Orange-brown precipitate N o change Sulfanilamide Greeniah-blue precipitate Blue precipitate Blue sediment Colorless aupernatant No change Yellow-green precipitate Sulfapyrazine Gray-green precipitate No change Yellow precipitate Sulfapyridine Liqht green precipitate Bright green precipitate Gray-green precipitate Light yellow-brown precipitate Light brown precipitate No change Yellow-green precipitate Gulfssuxidine Gray-green precipitate Gray precipitate Gray sediment Cloudy supernatant} Gray sediment Faint amber aupernatant} No change Sulfathiazole Greenish-brown precipitate Brownish-purple precipitate Pur le precipitate D t violet-purple precipitate Noa rchange
Sulfadiazine
]
}
151
for Observed
Change to Appear Immediate 5 seconds 10 seconds 5-10 minutea 30 minutes Immediate 5 seconds 15 seconds 30 seconds 1 minute 2 minutes 10 minute8 15 minutes 30 minutes Immediate 15 seconds 1 minute 3 minutes 5 minutes 15 minutea 20 minutes 30 minutes Immediate 5 seconds 2 minutes 30 minutes Immediate 10 seconds 5 minutes 30 minutes Immediate 230seconds minutes Immediate 1 second 10 aeoonds 1 minute 5 minutes 15 minutes 30 minutes Immediate 5 seconds 30 ieoonds 2 minutea 20 minutea 30 minutes
Immediate 15 seconds 1 . 5 minutea 5 minutea 30 minut-
INDUSTRIAL AND ENGINEERING CHEMISTRY
152
Table II. Cobaltous Ion-Sulfonamide Color Changes Sulfonamide Sulfacetimide Sulfadiazine
Sulfamerazine
Observed Changes in Order of Appearance Dilution of reagent, no reaction K O change Deep pink-red solution Same with gelatinous precipitate Clear gelatinous sediment Pink-red supernatant (precipitate m a y ] not appear) No change Light pink-white precipitate Rose, clear solution Same with gelatinous precipitate Pink gelatinous sediment Rose supernatant KO change Pink-white precipitate Light violet-white precipitate KO change Pinkish-blue precipitate Light blue gelatinous recipitate Light blue sediment Colorless supernatant No change Amber solution Salmon-pink solution Same with gelatinous precipitate Pink gelatinous sediment Light amber-pink supernatant} Bluish-pink sediment Cloudy supernatant i’io change Pinkish-white precipithte Light violet-pink precipitate Pink-white precipitate Pink-white sediment Pink supernatant K O change Rose solution N o change Pinkish-blue preoi,pitate Vlolet-blue precipitate Violet-blue sediment Lavender supernatant} K O change
}
Sulfamethazine Sulfanilamide
Sulfapyrazine
P
}
Sulfapyridine
}
Sulfasuxidine Sulfathiazole
Total Time for Observed Change t o Appear Immediate 30 minutes Immediate 5 seconds 5 minutes
30 minutes Immediate 10 seconds 15 seconds 10 minutes 30 minutes Immediate 10 seconds 30 minutes Immediate 30 seconds 5 minutes 30 minutes Immediate 2 seconds 5 seconds 5 minutes 10-20 minutes 30 minutes
Immediate 30 seconds 3 minutes 5 minutes 30 minutes Immediate 30 minutes Immediate 10 seconds 2 minutes 30 minutes
Modifications of the tests for special problems have been considered, but they are so obvious and instances so varied that details will not be given here. In some cases, mixtures can be separated by fractional precipitation or solution and the components identified. In such cases, it may be desirable to confirm results of these tests by means of chemical and physical properties of the substances. Calamari et aE. (3) have studied and summarized the chemical and physical properties of some of the most important sulfonamides for purposes of identification.
Vol. 17, No. 3
The blue precipitate formed when cupric sulfate is added t o 0.1 N sodium hydroxide should be obseked as a precaution against mistaking it as a positive copper-sulfanilamide test. This will, of course, be obtained if excess sodium hydroxide is present in the filtrate being examined, so that it is very important to take more unknown material than can be dissolved in the 5 ml. of alkali used for the test. If the unknown happens to be an alkali-insoluble material, such as sulfaguanidine, a questionable precipitate will result; this can be avoided by adding the unknown to the alkali in small amounts and noting whether or not it dissolves. Alkaliinsoluble sulfonamides-e.g., sulfaguanidine-can be differentiated and identified by tests such as those of Calamari et al. ( 3 ) . Another test for checking a positive copper-sulfanilamide reaction may be performed aa follows: To the reaction mixture containing the precipitate are added 5 ml. of 30% sodium hydroxide followed by 1 ml. of 5% potassium cyanide. The coppersulfanilamide and copper hydroxide precipitates both dissolve in strong sodium hydroxide to give blue solutions. On the addition of potassium cyanide, such a blue solution resulting from sulfanilamide turns yellow to amber, while one resulting from copper hydroxide is decolorized. Both cobalt and copper reaction products of other sulfonamides react similarly to sulfanilamide with this cyanide test. Arreguine ( d ) reports that sulfanilamide can be detected in the presence of its substitution derivatives by the indophenol reaction, using thymol in ammoniacal medium. Cobalt-ammonia-sulfonamide complexes have been studied, and although they might be used in supplementing information obtained from tests reported here, they are not so specific and are not considered necessary for inclusion in this paper. Cerium, chromium, iron (ferric, ferrous, ferricyanide, ferrocyanide), manganese, mercury, nickel, and silver salts were tested in a search for other metals which would give easily differentiated sulfonamide reactions such as those of cobalt and copper, but they were found unsatisfactory. Sodium sulfanilamide with cerium nitrate yields a precipitate of large, clear plates which might be useful for microscopic identification. LITERATURE CITED
(1) American Medical Association, Council on Pharmacy and Chemistry, J . Am. Med. Assoc., 118, 730 ( 1 9 4 2 ) . (2) Arreguine, V., Anales asoc. Q U ~ argentina, . 31, 38 (1943). (3) Calamari, J. A,, Hubata, R., and Roth, P. B., IND. END.CHEM., ANAL.ED.,14, 5 3 4 (1942). (4) Lott, W. A., and Bergeim, F. H., J . d m . Chem. Soc., 61, 3 5 9 3 (1939).
(5) U.S. Pharmacopoeia, 1 2 t h Revision, 1942.
Method for Classification of Petroleum Waxes A N T H O N Y KINSEL AND JOSEPH PHILLIPS Petrolia Laboratory, L. Sonneborn Sons, Inc., New York, N. Y.
N
EW petroleum waxes are constantly appearing on the market, owing to the demand of our Armed Forces and the fact that imported waxes are very scarce or, in some cases, unobtainable. Petroleum waxes have in the past been grouped into two broad classes-crystalline (or paraffin) and amorphous (or microcrystalline). This terminology is now obsolete but is still used to a certain extent by the industry, although it is generally understood that truly amorphous waxes do not exist. The two classes of wax have widely divergent applicabilities, and they have been differentiated by assuming that crystalline waxes are hard and brittle, and that the so-called amorphous waxes are soft and plastic. That this is not necessarily true was indicated by Marcusson and Schlutter (8),who showed that ceresine wax (which is hard and dry) is the same as amorphous wax of petro-
leum. In general, waxes of low molecular weight are more crystalline than those of high molecular weight. Crystallographic analysis does not differentiate clearly between the two types of waxes. Padgett, Hefley, and Henrikson (11) have shown that all forms of waxes occurring in refinery practice, whether crystalline or amorphous, are sufficiently impure to give needle or foliaceous crystals. On the other hand, Buchler and Graves (I) have shown thatwhenwaxes of low or high melting points, molecular weights, etc., are sufficiently purified, they crystallize in plates. Small amounts of impurities change the plate form to needle form. Finally, Gurwitsch ( 6 ) states that both natural and artificial petrolatums show a microcrystalline character under the microscope in polarized light. Possibly this was the reason why the petroleum industry aban-
