where (j), from which work it is apparent that mixtures of the glycols are more amenable to qualitative and quantitative analysis than are their dibenzoates, because the spectra of the lattrr are extremely similar. ACKNOWLEDGMENT
IT.H. T. Davison is thanked for the initiation and direction of the early part of this work. Thanks are also due to Kilfred Cooper and his colleagues for providing most of the samples of polyurethanes examined and to the Dunlop Rubber cO., Ltd*,for permission to publish this paper.
LITERATURE CITED
(1) Bayer, O., Muller, E., Petersen, S., Piepenbrink, F.7 ivindemuth, E., Rubber Chem. and 2'~chnol. 23, 812 (1950); Angew. Chem. 62,57 (1950). (2) Colthup, N. B., J . Opt. Sac. Am. 40, 397 (1950). (3) Corish, P. J., M.Sc. thesis, university of Wales, 1956. (4) Corish, P. J., Davison, W. H. T., J . Chem. Sac. 1955, 2431. (5) Corish, P. J., Davison, W. H. T., work. (6)1955, DaVison, 3270, W. H. T., J . C'hem. SOc. (7) Davison, IFr. H. T., Corish, P. J., Ibid., 1955, 2428. ( 8 ) Davison, w.H. T., Corish, p. J.1 unpublished work. (9) Dombrown, B. A., "Polyurethanes," Reinhold, New York, 1957.
(10) Dunn, J. P., Rubber World 137, 419 (1957). (11) Dyer, Elizabeth, Bartels, G. W., J . Am. Chem. SOC.76,591 (1954). (12) Haslam, J., Analyst 76, 33 (1951). (13) Hill, F. B., Young, C. A., Nelson, J. A,, Arnold, H. G., Ind. Eng. Chem. 48,927 (1956). (14) Marvel, C. S., Young, C. H., J . Am. Chem. sot. 73, 1066 (1951). (15) Muller, E., Bayer, O., Petersen, S., Piepenbrink, H. F., Schmidt, F., \Veinbrenner, E., Rubber Chem. and Technol. 26, 493 (1953); Angew. Chem. 64, 523 (1952). (16) Seeger, N. V.,hlastin, T. G., Fauser, E. E., Farson, F. S., Finelli, A. F., Sinclair, A . E., Rubber Chem. and Technol. 27, 430 (1954); Ind. Eng. Chem. 45, 2838 (1953). RECEIVEDfor review October 13, 1958. Accepted April 1, 1959.
Spectrophotometric Determination of p-Aminosalicylic Acid, rn-Aminophenol, and p-Aminophenol P. A. CACCIA Veterans Administration Hospital, Sunmount, N. Y.
b Sodium nitropentacyanocobaltate(11) reacts with aromatic compounds having NH2 and OH groups meta to one another to give an orange compound that can be determined spectrophotometrically. The reaction was carried out with p-aminosalicylic acid and m-aminopkenol; 0- and p-aminopt.enol gave no reaction. The colored compound obtained by reacting the cobaltate(l1) reagent and p-aminosalicylic acid was obtained in crystalline form, and its structural formula and molecular weight were determined. A method is given for analyzing mixtures of aromatic compounds having NH2 and OH groups in the ortho, meta, and para positions; this general method includes the determination of the para isomer with 8quinolinol.
T
patients are treated simultaneously with p-aminosalicylic acid and isonicotinic acid hydrazide. but these cannot be determined indt.pendently in blood or urine because both compounds give the same reaction ( 5 , 8, 9). This difficulty is also encountered a i t h other mixtures of primary amines. -1 recent spectrophotometric determination of p-aminosalicylic acid in blood and urine (3) takes advantage of the specific reaction of a new reagent, scdium nitropentacyanocobaltate(I1) [hereafter called cobaltate(I1) reagent], \yith SHa and OH groups in the meta I-BERCULOSIS
1306
ANALYTICAL CHEMISTRY
position. The reaction takes place in a slightly acid medium (pH 4.6 to 5 ) to give an orange complex that can be measured spectrophotometrically a t 440 mp. The color reaches its maximum intensity after 1 hour. The absorbance conforms to Beer's law up to 7 5 y per ml.. x i t h a deviation from 7 5 to 125 y per nil. m-Aminophenol undergoes the same reaction n i t h cobaltate reagent. Purified o-aminophenol gave no color reaction with the cobaltate reagent. p-Aminophenol was difficult to purify. also, an aqueous solution containing 100 y of this compound per ml. acquired a yellon color on standing a t room temperature for a few minutes. Therefore, in order to analyze mixtures of the three aminophenol isomers, a spectrophotometric method based on the reaction of p-aniinophenol and 8-quinolinol was developed. These two spectrophotometric methods can be combined with any general method based on diazotization and coupling to analyze a mixture of primary amines for the meta, ortho, and para isomers. For example, in a mixture of m-, 0-,and p-aminophenol, the total amount of the isomers is determined by diazotization and coupling. m-Aminophenol is then determined with the cobaltate reagent; p-aminophenol, with 8-quinolinol; and o-aminophenol, by difference. EXPERIMENTAL
of Color Reagent. nitropentacyanocobaltate(1V)
Preparation
Sodium
was prepared and used in the same way as the iron salt, sodium nitroferricyanide, except that cobalt was substituted for iron (4). The reagent should be prepared under a hood. A solution of 120 grams of sodium nitrite in 180 ml. of water IT-as added slowly to a solution containing 30 grams of cobalt nitrate hexahydrate in 50 ml. of water and 15 ml. of glacial acetic acid. Then 18 grams of sodium cyanide M as added and the temperature was brought to about 60" C. during mixing. The solution was then concentrated a t a low temperature until crystals of sodium nitropentacyanocobaltate(IV), Sa2Co(CN)&02, were formed. The crystals should be stored in a brown bottle. The color reagent, sodium nitropentacyanocobaltate (11) [iYa&o (CY)& 0 2 ] , was prepared by mixing equal volumes of a 47, aqueous solution of the sodium nitropentacyaiiocobaltate(1V) and 4h' sodium hydroxide. This reagent is unstable and should be discarded after every determination. Determination of Sodium p A m i n o salicylate.
STAXDARD SOLUTIOS.An
amount of the dihydrate salt equivalent to 100 mg. of sodium p-aminosalicylate was dissolved in water and diluted to 100 ml. By diluting 10 ml. of this stock standard to 100 ml., a working standard was obtained containing 100 y of sodium p-aminosalicylate per ml. Standard Curve. I n t o a set of cuvettes was placed 5 , 10, 25, 50, 7 5 , 100, and 125 y of sodium p-aminosalicylate; enough water was added to each cuvette to bring the volume to 1 ml. Then 1 ml. of freshly prepared cobaltate(11) reagent and 1 ml. of 3 5 acetic acid were added to each cuvette, and the solutions m r e mixed. The foregoing
:mounts gave a final p H of about 5 , Any mineral acid can be used in place of acetic acid. The solutions were alloned to stand a t room temperature for 1 hour. Then 7 ml. of water was added to each cuvette, the solutions were mixed, and the absorbance n a s measured on a spectrophotometer a t 440 mp after the blank had been set a t zero absorbance. The blank containing the same ingredients except the p-aminosalicylic acid a as colorless. The absorbance follow Beer's law u p to 75 y per ml. The color is stable for many hours after it reaches maximum intensity; after standing 24 hours a t room temperature, the colored complex loses only about 10% of its intensity. Crystallization of Colored Compound. T h e colored compound obtained f i o m the reaction of p-aminosalicylic acid and the cobaltate reagent was crystallized in the folloir ing manner:
A solution n as prepared Containing 1. i 5 grams of sodium p-aminosalicylate in 10 nil. of water. Then 2.66 grams of the sodium nitropentacyanocobaltate(IV) crystals was added and the solution \\as mixed. Sodium hydroxide (0.6 gram) was dissolved in 20 ml. of 95% ethyl alcohol by heating, and this was added to the first solution. While the solution was still warm from the hydroxide addition, enough glacial acetic acid 11as added to bring the p H to about 5 . The solution was allowed to stand in a stoppered bottle at room temperature while the crystals separated out. It was filtered under vacuum and washed with absolute alcohol and then with ether. The crystals thus obtained were dark bro\$n and very soluble in water, to ~ h i c ha few micrograms imparted an orange color. Molecular Weight Determination. T h e quantitative determination of nitrogen shon s t h a t only one amine and 1 mole of p-aminosalicylic acid take part in t h e reaction. T h e Folin\Vu digestion, followed by Sesslerization, gives 2.887, nitrogen; the Kjeldah1 method gives 2.97%. For each nitrogen determination 10 mg. of the product was used. A Kjeldahl determination of the sodium nitropentacyanocobaltate(1V) gives negative results for nitrogen. During digestion the only nitrogen fiwd as ammonium sulfate IS the nitrogen oi the amine. The formula of the new compound thus could be n ritten [CsH3COOT\"OSOCoI C S ) ~ ] S ~The ~. exact molecular neight of the new commund. calculated from the structural formula. 1s 461.118. The theoretical values are close to those determined by analysis: Y
S,
co Sa
Per Cent Theory Euptl. 3 03 2 97 12 78 12 31 18 92 20 36
Structural Formula. T h e groups of p-aminosalicylic acid t h a t react t o form the colored compound are t h e S H z and OH in meta position. The cyanide group does not take part in the reaction, as Baudisch ( 1 ) shows in the reaction of sodium ferricyanide with an aromatic compound having a n NO group. S a a F e ( C N ) Z RSO + Na3Fe(CT\;)sRS0 X. Feigl and coworkers ( 6 ) also shon that the cyanide group does not take part in the reaction. The S O 2 is then the only reactive group of the color reagent. The nitrogen bonds with the amine and with the hydroxyl, with one oxygen of the S O 2 combining with one hydrogen of the amine and n i t h one hydrogen of the hydroxyl to form water:
+
+
10 minutes or between 20 aiid 30 minutes, because the color begins to fade after 30 minutes. The absorbance conforms to Beer's law over the range studied; the color is faint blue with 1 y and deep blue with 10 y of p-aminophenol. DISCUSSION
p-riminophenol can coiubiiie n-ith a base because of the acidic phenolic hydroxyl and with an acid because of the amine. Oxine (8-quinolinol) has the structural formula,
OH
The quinoidal form oi thi. qtructure is
0
-"\d-
--n---s/-
+H,O
The reaction and the structural formula of the colored compound are probably :
and in water solution
COOXa
sii
equilibrium
1-
I
Y
SH,
AS&
+ CH7COOSa + H20 The ring structure.
=c-0 I
0
, is
formed only nhen S H , aiid OH are meta to one another. Determination of p-Aminophenol. T h e color reagent was prepared by dissolTing 100 mg. of 8-quinolinol in 1 ml. of glacial acetic and diluting t o 10 nil. a i t h 11-ater. Sodium hydroxide, 0.lAl7. Standard Solution. d 10-nig. quantity of purified p-aniinophwol was dissolved in n a t w and diluted to 100 ml. By diluting 10 nil. of this stock standard to 100 ml. n ith water, a working standard containing 10 y of p-aminophenol per ml. was obtained. Standard Curve. I n t o a set of cuwttes was placed 1 to 10 y of paminophenol in 1-y increments. Enough water was added to each to make a 1-ml. volume. Then 1 nil. of 8-quinolinol was placed in c w h cuvette. A blank was prepared a t the same time containing 1 nil. of water and 1 ml. of 8-quinolinol; it n-as colorless. The solutions werp alloned to stand a t room temperature for 5 minutes, after which 2 nil. of 0.l.V sodium hydroxide nas added to each cuvette and the solutions were mixed. I n 20 minutes the absorbance was measured a t 620 nip after the blank had been set at zero absorbance. Measurements should be made within
ma\ be established beta ecii the tautomeric forms. Berg and Beckw ( 2 ) have shon-n that the tlro form> ran br part of the same compound as i n i n r h i n e S
RE-4GEh-Ts.
0
OH
8-Quinolinol ha. an acidic. character because of its phenolic component and a basic character becausc of its pyridine component. p-.lniinophenol and the two forms of the oyiiie could be part of the same compound. When the phenolic component react* with the amine and the p j ridine component reacts with the hydroxyl of the p-aminophenol, the product of thta reaction might have the following -tructural forniula :
o-Ainiinophenol.~,~-ai~iino~)licnol. and p-aminosalicylic acid gin, no color rcaction with 8-quinolinol. Gilnian ( 7 ) states that i n disubstituted aromatic derivatives of the type VOL. 31, NO. 8, AUGUST 1959
1307
A-C6H4-B, the ortho and para compounds will have a vinylogous relationship to the system A-B, but the meta isomer will not be a vinylog of -4-B. It is significant that aromatic systems containing hydroxyl and amino substituents in an ortho or para relationship are among the most powerful autooxidation inhibitors, whereas aromatic compounds containing KHz and OH in meta position are not oxidation inhibitors. I n the organic types the discordant system can be relieved by tautomerism of a sort that is different from that of the parent structure:
Aromatic systems containing hydroxyl and amino substituents in ortho or para relationship change into the quinone or the imino form. The structural form of p-aminosalicylic acid or of m-aminophenol shows that the KH2 and OH cannot change because the meta isomer is not a vinylog of 8-B. Therefore, only when the NH2 and O H are in meta position can they react with cobaltate, and in a mixture of aromatic compounds, only those containing the amino and hydroxyl substituents in a meta relationship will give the reaction with sodium nitropentacyanocobaltate-
LITERATURE CITED
(1) Baudisch, O., Ber. 5 4 , 413 (1921). ( 2 ) Berg, R., Becker, E., Zbid., 73-172 (1940); 2. anal. Chem. 119, 81 (1940). (3) Caccin, P. .$., Ant. Rev. Tuberc. Pulmonary Diseases 7 5 , 105 (1957). (4) Zbid., 7 6 , 1071 (1957). (5) Deeb, E. N., Vitagliano, G. R., J . Am. Pharm. Assoc. 4 4 , 182 (1955). (6) Feigl, Fritz, “Chemistry of Specific,
Selective and Sensitive Reactions,”
p. 3i7, -4cademic Press, New Pork, 1949 _ _ _.
( 7 ) Gilman, H., “Organic Chemistry,” pp. 1909-28, Kiley, New York, 1947. (8) Klyne, W . j Kewhouse, J. P., Lancet 2, 611 (1948). (9) Marshall, E. K., Jr., Proc. SOC.Ezptl. Bid. X e d . 68, 471 (1948).
RECEIVEDfor review June 7, .4ccepted April 3, 1959.
1958.
Ident ificat io n of Crysta IIine Ferroce nes by X-Ray Diffraction WILLIAM L. BAUN Materials laboratory, Wright Air Development Center, Wright-Patterson Air Force Base, Ohio
b X-ray diffraction patterns for 22 polycrystalline ferrocenes are presented. These compounds are of interest because of their current and potential applications. They are particularly well suited for x-ray diffraction analysis because of their crystalline nature and the individualistic patterns obtained.
B
Table
I.
Comparison of Powder Data Intensities from Ferrocene
III, d , A. 5.83 5.10 4.85
Flat specimen
DehyeScherrer camera
100 40 10
100 70 40
5
15 20
IS(C Y C L O P E N T A D I E N Y L ) I R O N (11)
compounds, commonly known as ferrocenes (so named to emphasize the analogy to benzene) have aroused considerable interest since the discovery of ferrocene eight years ago (4). I n this time over 100 publications have appeared concerning metal-cyclopentadienyl compounds. The applications and potential uses of these compounds depend mainly on their behavior as reducing agents, antioxidants, and as excellent organic carriers of iron in high concentration (5). Accordingly, a method of qualitative and quantitative analysis is necessary for these compounds. Infrared absorption analysis in the rock salt region, used for organic syntheses, has certain limitations in ferrocene derivative detection, differentiation, and quantitative analysis (1). From the present data, i t appears that x-ray diffraction is not seriously limited for the analysis of the solid crystalline members of the ferrocene family.
1308
ANALYTICAL CHEMISTRY
3.32 3.06
2
MATERIALS A N D APPARATUS
Samples of ferrocene derivatives were prepared largely by the Organic Synthesis Section of the Organic Materials Branch, Materials Laboratory, JTright Air Development Center. The compounds were recrystallized from various solvents. Their synthesis and crystallization with experimental data have been detailed by Rausch, Vogel, and Rosenberg (6-8). A chemical analysis was run on each sample and its melting point determined; each sample was very pure. Sample preparation for x-ray diffraction analysis consisted of grinding the sample in a boron carbide mortar and placing the sample on a ground-glass slide. This slide method of mounting the sample, which undoubtedly introduced some preferred orientation and subsequent inaccuracies in intensities
was necessitated by the small amount of pure sample available. Preferred orientation effects did not seem to affect reproducibility of the same samples, because samples could be reprepared over and over without appreciable change in relative intensities. However, a comparison between flat specimen difTractometer and Debye-Scherrer camera intensities showed that intensities from the flat specimen did not originate from a random powder sample. Table I s h o m this comparison for the first 10 lines of the parent compound, ferrocene. All flat specimen powder patterns were run a t room temperature utilizing a Sorelco diffractometer (Xorth American Phillips Co., Inc.) equipped with scintillation counter and pulse height analyzer. Kickel-filtered copper radiation was used, with the pulse height analyzer used to decrease background from iron fluorescence. Vanadiumfiltered chromium radiation and a 114.6mm. Debye-Scherrer camera were used to obtain the intensities shown in Table I. DISCUSSION
X-ray diffraction appears ideally suited to the analysis of ferrocene derivatives and should prove valuable especially where chemical similarities make detection and quantitative analysis of these compounds difficult by infrared absorption techniques. Figure 1 illustrates the problems encountered in the analysis of ferrocene derivatives, showing the infrared absorption spectra in potassium bromide from 2 to 15
