ment, and computing the cystine concentration is within 30 seconds. Effects of temperature, pH, and potential interferences in this reaction have been discussed. This work was done a t a temperature of 25.0 =t0.1’ C. and a t p H 5.8. Although the measurement method has been developed and d e w i b e d for the potentiometric detection of the rate of a chemical reaction i t should be applicable
to other signal systems. Also the method should be applicable to systems with nonlinear response curves. These possibilities are being investigated.
tists,” p. 356, W. A. Benjamin, New
LITERATURE CITED
(3)York, Malmstadt, 1962. H. v., Hicks, G. p., A ~ cHEM. ~ 32, ~ 3g4. (1960). (4)Malmstadt, H. T., Pardue, H. L.. Ibid., 33, 1040 (1961). ( 5 ) Pardue, H. L., Shepherd, S. A., Zbid., 3 5 , 2 1 (1963).
( 1 ) BlaedeI, W. J., Hicks, G. P., ANAL. CHEW34, 388 (1962). ( 2 ) Malmstadt, H. Y,, Enke, C. .G., Toren, E. C., “Electronics for Scien-
RECEIVEDfor review October 3, 1963. Accepted Xovember 29, 1963. Research supported in part by research grant from the National Institutes of Health, USPHS.
Spectrophotometric Determination of p-Phenylenediamines and p-Aminophenols with Ninhydrin ROBERT SUFFIS, ADELE LEVY, and DONALD E. DEAN Shulton, Inc., Clifton, N. J.
b The use of ninhydrin as a spectrophotometric reagent for the determination of low concentrations of p phenylenediamines and p aminophenols is proposed. Under the prescribed conditions no color is produced by any of the other aromatic amines investigated. This method is applicable to the determination of p-phenylenediamines and p-aminophenols in the presence of their ortho and meta isomers. Synthetic samples in the concentration range 0.05 to 0.50% were prepared and analyzed by this technique. The results had a range of i10% from the mean.
-
N
-
has found wide utility as a reagent for the spectrophotometric determination of amino acids as well as primary and secondary aliphatic amines. However, there appears t o be no published \T-ork concerning the quantitative spectrophotometric determination of aromatic amines with this reagent. Aromatic amines are known to give colored condensation products with ninhydrin. Most of these compounds have a color intensity so low that the reaction is uqable only as a spot test and then only if the aromatic amine is present in high concentration. Of the compounds investigated only p-phenylenediamines and p aminophenols give colors sufficiently intense for the spectrophotometric determination of trace amounts. Sinhydrin has been known to react with aliphatic primary amines and amino acids since its discovery by Ruhemann in 1910 ( 4 ) . The product of this reaction is a compound known as Ruhemann’s purple, which has an absorption maximum a t 570 mp. d reproducible spectrophotometric method based on this test was developed for the deter-
636
IXHYDRIN
ANALYTICAL CHEMISTRY
mination of amino acids by Moore and Stein ( 2 ) . The reaction of ninhydrin with aniline and its derivatives was first studied by Ruhemann ( 5 ) and later by Moubasher ( 3 ) . The reaction product that forms with most aromatic amines is compound I.
Ortho- and m-aminophenols and phenylenediamines did not give the highly colored reaction products produced from the para compounds. A possible reason for the reactivity of the para derivatives is given below. ;1Schiff base (111) may form from compound I by the loss of a mole of water. This compound can exist in a stable quinoid form (IV).
I Aniline forms a different product than the substituted anilines investigated. I n this case 2 moles of aniline react with 1 mole of ninhydrin t o give compound 11.
0
I1 These reactions have been utilized by Barakat, Wahba, and El Sadr (1) for the qualitative identification of aromatic amines. When 10 mg. of aromatic amine were allowed to react with 10 mg. of ninhydrin in 2 to 3 ml. of solution, colors were produced in most cases. This study was undertaken to apply the method of Moore and Stein to the quantitative determination of aromatic amines, Gnder these conditions most aniline derivatives gave a color that was not significantly different from that of a reagent blank. The products formed had molar absorptivities less than 20. The only compounds that gave more intense colors were p-phenylenediamines and p-aminophenols, which gave blue or purple products with molar absorptivities of 760 t o 9000 (Table I).
The intense color of the p-phenylenediamine or p-aminophenol adduct with ninhydrin may be accounted for by the high degree of stability which an amino or hydroxy group imparts to the quinoid form. The quinoid form would be expected to absorb light a t a longer wavelength with a greater intensity than the nonquinoid form. The meta derivatives have a much lower color intensity, because a quinoid form is not possible. Ruhemann (6) described the reaction of o-phenylenediamine with ninhydrin to give a yellow quinoxaline (Ir).
The absence of color in the reaction of o-aminophenol with ninhydrin may be explained by the added stability of the noriquinoid form (VI) due to hydrogen bonding.
Table I.
Study of Reaction Conditions for Color Development Reactions
Heating period
0
15 min. 30 min. 1 hour 2 hours
8640 8430 8720 4250
Molar absorptivity Time elapsed after color development 15 min. 1 hour 2 hours p-Phenylenediamine 8640 8600 8410 8400 8700 8640 ... ...
8450 8370 8520
Table II.
Reaction of Aromatic Amines with Ninhydrin
Absorption Reac- maxition mum wjth (wave- Molar nin- length), absorphydrin mp tivity
4 hours 8130 8040 8310
Aminophenol m-Aminophenol 555 1640 p-Aminophenol p-Aminophenol o-Phenylenedi15 min. 1640 1620 1610 1580 1490 amine 1580 1580 1580 1560 1410 30 min. m-Phenylenedi1 hour 1710 1700 1670 1670 1580 amine 2 hours 1350 ... ... ... ... p-Phenylenediamine 540 8640 Aniline p-Aminobenzoic acid p-Bromaniline the same procedure. Its concentration p-Sitroaniline was determined by comparing its abp-Aminodisorbance with the calibration curve. 572 3700 phenylamine DETERMINATION OF p-AYINOPHENOL. o-Nitro-p-amino530 770 phenol This procedure is identical with that p-Toluenedifor p-phenylenediamine except for two 555 3400 amine modifications. The standard solution o-Nitro-pshould have a concentration of 4 mg. This paper describes the utilization phenyleneof p-aminophenol per ml. and the final diamine of the ninhydrin reaction as a specific at 555 mp. absorbance reading is 4,6-Dinitro-2method for the quantitative deteraminophenol mination of small amounts of p-phenylRESULTS AND DISCUSSION enediamine and p-aminophenol in the presence of other aromatic amines. A study was made of the reaction Table 111. Determination of p-Phenylconditions required for color developenediamine and p-Aminophenol ment with p-aminophenol and p-phenylEXPERIMENTAL ReReenediamine. Table I indicates t h a t for Added, covered, covery, Reagents and Apparatus. Citrate heating times between 15 minutes and 1 70 70 70 buffer, 21.008 grams of citric acid hour molar absorptivity is reasonably p-Phenylene0.50 0.47 94 plus 200 nil. of 0.1-1' sodium hydroxFor constant for both reactions. diamine" 0.50 0.49 98 ide, was diluted to 500 ml. 0.25 0.28 112 heating periods of 2 hours there is a Potassium cyanide reagent, 5 ml. of 0.25 0.23 92 marked decrease in molar absorptivity. 0.OlJP potassium cyanide, was diluted 0.10 0.093 93 The color produced is stable for 1 0,102 102 to 250 ml. with methyl Cellosolve. 0.10 hour, after which there is a slight de0.05 0.054 108 Xinhydrin reagent, 250 ml. of po104 0.05 0.052 crease in intensity. T o get the best tassium cyanide reagent, was mixed possible precision, heating for 15 with 50 ml. of 501, solition of ninhydrin p-Amino0.50 0.52 104 in methyl Cellosolve. minutes and measuring the absorbance phenolb 0.50 0.45 90 Beckman DK-2 spectrophotometer. 0.25 0.28 112 immediately after color development n.25 0.26 io4 Procedure. QUAI~ITATIVE~ A L - have been utilized. 108 0.10 0.108 YSIS. One milliliter of a sample conTable I1 indicates t h a t the ninhydrin 0.103 103 0.10 taining 10 mg. of aromatic amine was method can be used for the quantitative 96 0.05 0.048 added to 5.0 ml. of titrate buffer in a 0,057 114 0.05 determination of p-phenylenediamine test tube. Siu milliliters of ninhydrin and p-aminophenol. The range is reagent were added and the mixture a Each sample contained same amount approximately =!=IO% from the mean. was heated a t 100' C'. for 15 minutes, of 0- and ni-phenylenediamine. Each sample contained same amount then cooled under t a p water and diBecause of variations in color developof o- and m-aminophenol. luted t o 25 ml. in a volumetric flask. ment, it is necessary t o run standards T h e visible spectrum of the sample with each set of analyses. was measured in a 1-cm. cell by t h e The data shown in Table I11 indicate Beckman DK-2 spectrophotometer. the specific nature of the ninhydrin reDETERhIINATIOK O F PHENYLENEenediamine and p-aminophenol in the action with aromatic amines. Of the DIAMIKE. A\liquots oj 0.0, 0.2, 0.4, 0.6, presence of their ortho and meta 15 compounds investigated, only p0.8, and 1.0 ml. of a freshly prepared isomers. phenylenediamines and p-aminophenols standard solution containing 1 mg. of gave a positive test. Several substituted p-phenylenediamine per ml. were added LITERATURE CITED to six test tubes. Sufficient water was p-phenylenediamines and p-aminoadded to each to make a total volume (1) Barakat, M. Z., Wahba, S . , El Sadr, phenols, such as p-toluenediamine sulM. M., Analyst 79, 715-17 (1954). of 1.0 ml., then 0.5 ml. of citrate fate, o-nitro-p-aminophenol, and pStein. W. H.. J . Biol. Chem. (2) buffer and 1.2 ml. of ninhydrin reagent ~, ,Moore. S.. aminodiphenylamine, also give a blue 176,367'(1948). ' were added. The mixture was heated color with ninhydrin. Only nitro(3) Moubasher, R., J . Chem. SOC.1949, for 15 minutes a t 100' C., cooled, and _1238-9. - _ _ -. p-phenylenediamine does not give a diluted to 100 ml. The absorbance a t (4) Ruhemann, S., Ibid., 1910, 1438. test, probably because of the electron540 mp was immediately measured and ( 5 ) Zbid., p. 1449. attracting character of the nitro group, plotted against concentration. Csing the technique described we A 1.0-ml. aliquot cf an unknown pRECEIVED for review September 11: 1963. phenylenediamine solution was run by have been able to determine p-phenylAccepted December 11, 1963. ...
...
0-
+
+
+
+ +
VOL. 36, NO. 3, MARCH 1964
637
