Colorimetric Determination of Carbazole GERALD GILBERT, R. M. STICKEL, and H.
H. MORGAN, Jr.
Applied Research laboratory, United States Steel Corp., Monroeville, Pa.
b A colorimetric method for determining carbazole has been developed, based on reaction with xanthydrol and glacial acetic acid in the presence of hydrochloric acid. The absorbance of the product is determined at 525 mp and the carbazole concentration is read from a standard curve previously prepared from pure carbazole. The relative precision of the method is within O.6Y0 and good agreement is obtained with infrared analyses. Evidence is presented to support the view that the colored product is a xanthylium salt, and a mechanism for its formation is postulated. The scope of the reaction is extended to include several substituted carbazoles, and several interfering compounds are listed.
A
simple method for the determination of carbazole in coal-tar fractions and other mixtures was required for a n investigation of the highboiling components of creosote. Of the numerous methods available, only the Kjeldahl nitrogen and infrared methods of analyses appear t o have widespread acceptance. Unfortunately, the Kjeldahl procedure will not distinguish between nitrogen compounds, and the infrared method, which is more selective, requires expensive equipment. Among the other methods reported in the literature are bromination (1) and treatment with formaldehyde (3). Colored products t h a t might be adapted to a spectrophotometric method have been reported in the reaction of carbazole with rhamnose ( 8 ) , vanillin and sulfuric acid (71, furfuraldehyde ( 4 ) , glyoxylic acid (4,nitric and sulfuric acids (9),and xanthydrol ( 2 ) . T h e last reaction, reported b y Arreguine (2), is adaptable t o a quantitatile colorimetric method. RAPID,
EXPERIMENTAL
Reagents. T h e xanthydrol reagent is prepared by dissolving 5 grams of Eastman Kodak Yellow Label xanthydrol in glacial acetic acid and making t h e solution t o a liter with glacial acetic acid. T h e hydrochloric acid reagent is prepared by diluting 50 ml. of concentrated hydrochloric acid t o a liter with glacial acetic acid. Procedure. A sample containing about 50 mg. of carbazole is weighed
accurately into a 100-ml. volumetric flask, dissolved in glacial acetic: acid with warming, if necessary, and diluted t o t h e mark with t h e acid. I n t o a 100-ml. volumetric flask are pipetted 1 ml. of sample solution, 5 ml. of xanthydrol reagent, and 5 ml. of hydrochloric acid reagent. The sides of the flask are then washed down with 10 ml. of glacial acetic acid. The solution is placed in a boiling water bath at 97' to 100 O C. for exactly 15 minutes, cooled to room temperature rapidly, and diluted to the mark with glacial acetic acid. The absorbance of this solution is determined at 525 mp and after correction for blank absorption, the amount of carbazole in the sample is determined from a standard curve, prepared from pure carbazole, which followed Beer's law. The color intensity will remain constant at room temperature even when the sample stands overnight, and therefore, any number of samples may be retained for colorimetric determination. The absorbance of the blank solution, prepared by the above procedure omitting the sample, is generally about 0.02. A high blank usually indicates incomplete cleaning of the volumetric flasks. Very small amounts of carbazole adhering to the equipment can markedly reduce the accuracy of the determination. A suitable cleaning procedure involves washing the volumetric flasks with acetone, rinsing thoroughly with water, allowing them to stand filled with a detergent solution for several hours, rinsing again with water, and then allowing them to dry. Apparatus. Both t h e Fisher Electrophotometer (with a green filter, 525 mp) and the Beckman DU spectrophotometer (at 525 mp) with 1-cm. cells were found satisfactory. Separate standard curves are required for each instrument. The absorption spectrum of the colored carbazole derivative was determined on a Cary recording spectrophotometer, Model 11-MS (Figure 1). The analytical measurements were made at 525 mp because this wave length is more accessible on the Fisher Electrophotometer than is the peak at 567 mp. The effects of heating time, amount of hydrochloric acid reagent, and amount of xanthydrol reagent are shown in Figures 2 and 3. Each point represents a separate experiment conducted under the above conditions \\ ith only the variation noted. DISCUSSION
Interferences. Compounds t h a t are
Table I.
Effect of Impurities on ColorForming Reaction
-
Carbazole Concentration, 5 09 x 10-6 G./Ml. Relatiye
Concn.,
error In
g./rnl. Absorb- carbazole
Impurity x None ... Acridine 7.87 Anthracene 5 . 1 0 Benzoic acid 5 . 7 2 Dibenzothio5.16 phene Diphenylene oxide 6.12 Naphthalene 5 . 0 7 2-Naphthol 5 . 7 8 Phenanthrene 5.30
ance
0.496 0.491 0.492 0.497
detn., yo ... -1.0 -0.8 +O. 2
0.492
-0.8
0.493 0.491 0.525
-1.0
+6.0
0.495
-0.2
-0.6
Table II. Absorption of Carbazole Derivatives and Indole
Concn., G . / M . x hbsorbance, 525Mp
Compound N-Acetylcarbazole N-Cyanoethylcarbazole Dodecahydrocarbazole Indole 2-Methylcarbazole N-Methylcarbaaole
8.39
0.05
6.4
0.369
6.05 5.20 4.95 3.38
0.0 0.474 0.299 0.241
representative of those often associated with carbazole and of a variety of other functional groups were added to t h e reaction mixture t o determine their effect on t h e absorbance. T h e d a t a in Table I indicate t h a t the substances common t o carbazole fractions obtained from coal tar, such as anthracene, phenanthrene, acridine, and dibenzothiophene, show practically no interference with the colorimetric determination, even when present in approximately equal concentration. Phenols such as 2-naphthol interfere more strongly but can be eliminated by extracting the sample n.ith aqueous sodium hydroxide. Several qualitative tests showed that small amounts of acetone will markedly reduce the intensity of the color when added either before or after the reaction. Structural Requirements for Reaction. T h e absorption of a number of available carbazole derivatives a t 525 mp after reaction with the xanthydrol reagents was determincd to elucidate further the nature of the reaction. T o the data in Table 11 may be added the VOL. 31, NO. 12, DECEMBER 1959
1981
WAVE
LENGTH, m i l l i m i c r o n i
Figure 1. Absorption spectrum of colored complex in visible region
Figure 2.
Sample concn., 0.963 mg. of carbazole per 100 ml. of solution
observation by Arreguine ( 2 ) that antipyrine and aryl semicarbazones also form the intense blue color. For carbazole, one is tempted to ascribe the color to reaction a t the amino group. However, the fact that the N-cyanoethyl and N-methylcarbazole derivatives also form the colored product indicates that the former reaction. if it does occur, does not lead to color formation. The data indicate that an aryl group susceptible to substitution by xanthydrol and conjugated with a nitrogen auxochrome is necessary for formation of the blue color and that a 9-arylxanthene (I) is probably formed. That this reaction is possible under the conditions of the present analysis is supported by reports of the xanthylation of many aromatic and heterocyclic compounds in acid solution (10).
Although no direct evidence is available, it is possible that these arylxanthenes formed from xanthydrol are converted to xanthylium salts, which are the colored final products shown below. This hypothesis is supported by the observation that the color disappears on neutralization and reappears on acidification, as is found with xanthylium salts in general. The hydrolysis of xanthylium salts t o colorless intermediates (usually ethers) and their
Figure 3.
method 10.5 35.0 59.6 65.2 87.7
98.5
1982
method 12.8 38.9 56.2 64.8 84.1 96.9
ANALYTICAL CHEMISTRY
Effect of reagent concentration on absorbance A. 6.
HCI reagent Xanthydrol reagent
reconversion with acid is also well known and was observed in the present case.
xanthydryl chloride in acetic acid oxidizes iodide ion to free iodine. Xanthylium salt (11) is analogous to the colored chloride hydrochloride formed in hydrochloric acid by S-phenylxnnthydrol and 9-phenyl xnnthydryl chloride (6).
I Table I l l . Comparison of Xanthydrol and Infrared Methods for Analysis of Carbazole Carbazole, % Infrared Xanthydrol
Effect of heating time on absorbance
'
I1
The conversion of the xanthene (I) to the colored xanthylium salt (11) is readily explained as a n oxidation by the xanthydryl chloride. The oxidizing nature of the latter may be demonstrated, for example, by the observation of Fosse (5), which the present authors have confirmed, that
Precision and Accuracy. T h e prese n t method has a relative precision better than 0.6%, which is the standard deviation found in several series of replicate determinations. T h e accuracy is similar to the infrared detmmination. It will depend largely on t h r nature and concentration of impurities present in the carbazole, as demonstrated in Tables I and 11. A comparison with an infrared method for determining carbazole in a number of samples derived from coal tar is shown in Table 111. The correlation coefficient for the two methods is 0.998. ACKNOWLEDGMENT
The authors thank C. E. Bole, J. T. Peters, H. A. Barnett, and C. R.
Manganaro of the Applied Research Laboratory, and C. S. Sheppard of the United States Steel Corp. Mellon Institute Project 237, for their advice and assistance during this work. LITERATURE CITED
(1) Ardashev, B. I., Trudy Ural. Ind.
Inst. im. 8. hf. Kirova 1938, s o . 6,
70-9. (2) Arreguine, Victor, Rev. univ. nacl. Cdjdoba ( A r g . )31, 1706 (1944). (3) CermBk, Miroslav, Chem. listy 45, 35-6 (1951). (4) Fleig, C., Compt. rend. SOC.biol. 6 5 , 283 (1908). (5) Fosse, R., Ann. chim. 6 , 13 (1916). (6) Gomberg, M., West, C. J., J . Am. Chem. SOC.34, 1529 (1912).
( 7 ) Haussler, E. P., Nitt. Gebiete Lebensm. Hyg. 38, 1 (1947). (8) Hayashi, Kaneo, J . SOC.7'rop. A g r . , Taihoku Imp. linio. 15, 20 (1943). (9) Sommer. L.. Chem. listu 47. 1415
(10) Wawzonek, S., in "Heterocyclic Compounds," R. C. Elderfield, Vol. 2, pp. 463-4, Wiley, New York, 1951. RECEIVED for review February 6, 1959. Accepted August 3, 1059.
Rapid and Specific Determination of Threonine MARTIN FLAVIN and CLARENCE SLAUGHTER Enzyme Section, National Heart Institute, Department of Health, Education, and Welfare, Bethesda, Md.
b The acetaldehyde liberated by periodate oxidation of threonine may b e directly measured, after reduction of excess periodate with a mercaptan, by the amount of dihydrodiphosphopyridine nucleotide (DPNH) oxidized by alcohol dehydrogenase. The procedure requires 5 minutes, as compared with 2 to 5 hours for previous methods. All of the required reagents are available commercially.
P
specific methods for the determination of threonine, particularly in mixtures with other amino acids, have involved the diffusion, or forced aeration, of the acetaldehyde formed by periodate oxidation into trspping reagents such as bisulfite ( 8 , 9 ) , p-hydroxybiphenyl (9), or dimedon ( 5 , s - dimethyl - 1,3 - cyclohexanedione) ( 5 ) . The acetaldehyde is then usually measured by color formation with phydroxybiphenyl (or gravimetrically, in the case of the dimedon derivative).. These methods require 2 to 5 hours, and in the bitter two cases have been found unreproducible. h'either has it been possible to repeat a procedure for the direct measurement of the acetaldehyde with dinitrophenylhydrazine ( 7 ) . Now, however, the acetaldehyde can be measured in situ with D P K H and alcohol dehydrogenase, provided excess periodate is first reduced with a mercaptan. The method has been reproducible during constant use throughout the past y f a r in connection with studies of the mechanism of the enzymatic formation of threonine ( 4 ) . REVIOUS
REAGENTS
Molar potassium phosphate, p H 7.5, and 40/,sodium metaperiodate (Mallinckrodt analytical reagent grade) are prepared in distilled water, as are all other reagents. A 10% aqueous solu-
tion, by volume, of 3-mercaptopropionic acid (Eastman h'o. 6270) adjusted to p H 6 with potassium hydroxide is made up weekly, as longer storage leads to a decline in sulfhydryl titer ( 3 ) . This and the following reagents are kept at 0" C. while in use, and stored frozen. D P X H (Sigma Chemical Co., disodium) is oreoared as a 0.0025M solution. o H adjusied to 7 . 5 . Twice-crystallized yeast alcohol dehvdroeenase was obtained from the NutriGonal Biochemical Co. as a suapension in ammonium sulfate of 60 mg. of protein per ml., and was stored at -20" C. Though i t assayed a t only 40,000 units per mg. (I), this preparation has been stable and entirely satisfactory for a year. For use, several milliliters of a 100-to-1 dilution are prepared in the following diluent: 0.1% bovine serum albumin, 0.01M reduced neutral glutathione, and 0.02M potassium pyrophosphate, p H 7.5. PROCEDURE
The sample, containing 0.02 to 0.1 pmole of threonine, is pipetted into a 1-ml. volume, 1-cm. light path silica cuvette, followed b y 0.1 ml. of phosphate buffer and enough distilled water for a final volume of 1.0 ml. Three cuvettes are run at one time, against a water blank. After adding 0.02 ml. of periodate to the reaction cuvettes, the solutions are mixed well and allowed to react for 30 seconds. (The oxidation of threonine is incomplete in 15 seconds.) Then 0.03 ml. of mercaptopropionate is added, and the mixture again stirred for 30 seconds. After adding 0.04 or 0.05 ml. of D P N H , the solutions are again stirred, and two successive absorbance readings are made a t 340 mp, in the Beckman D U spectrophotometer, at room temperature (25" C.). These will show no decline in absorbance if reduction of periodate t o iodide b y the mercaptan has been complete. Finally, 0.02 or 0.03 ml. of alcohol dehydrogenase is added, and the oxidation of D P N H by acetaldehyde is fol-
Table I.
Specificity of Assay for Threonine
Ile!rease in Absorb-
Compounds Added uL-Threonine ~~-Allothreonine 2,3-Butaiiediol DL-Serine DLThreonine DL-serine Glucose Gl!m?rol DL-Homoserine 0-Phosphohomoserine DL-Aspartate DL-Methionine Tartrate DIrThreonine tartrate
+
+
Amount, ance, pmole 340 M p 0 1 0.40 0 1 0 41 0 05 0 35 0 1 0 1 0 0 0 1
1 0 0 1
0 40 0
0 1
0
0 1
0 0 0
0 1 0 1 0 1
0 5 0 1 0 5
0 0 0.34
lowed a t 340 mp until two successive readings a t half-minute intervals show no further decline in absorbance. The total decrease in absorbance, corrected for dilution by the enzyme, is the measure of the amount of threonine present. The amount of enzyme is sufficient to bring the rraction to completion in 1 to 3 minutes. The use of much larger amounts xould reduce the specificity of the assay ( I ) . RESULTS AND DISCUSSION
The change in absorbance at 340 mp is strictly proportional to the amount of threonine added, from 0.01 to 0.1 pmole. An occasional reference cuvette should be run with a known amount of threonine for, although the absorbance change per 0.1 pmole of threonine rc,mains constant with any one batch of reagents, it has varied, with different batches, between limits of 0.40 and 0.47. These values are about one third less than would be predicted from the absorptivity, 6300, of D P N H a t 340 mp. The explanation VOL. 31,
NO. 12, DECEMBER I959
1983
