Spot Test Detection and Spectrophotometric Characterization and Determination of Carbazoles, Azo Dyes, Stilbenes, and Schiff Bases Application of 3-Methyl-2-benzothiazolone Hydrazone, p-Nitrosophenol, and Fluorometric Methods to the Determination of Carbazole in Air EUGENE SAWICKI, THOMAS R. HAUSER, THOMAS W. STANLEY, WALTER ELBERT, and FRANK
T. FOX
Roberf A. Taff Sanitary Engineering Cenfer, Cincinnati 26, Ohio
b Two new spectrophotometric methods for the determination of carbazole are introduced. Beer’s law was obeyed from 4 to more than 90 pg. of carbazole in the 3-methyl-2-benzothiazolone hydrazone and p-nitrosophenol procedures. Both methods can be used for the estimation of carbazole in the benzene extracts of airborne particulates. Two new spot tests for carbazole are also introduced. With the p-nitrosophenol test, 0.4 pg. of carbazole can be detected. In strongly alkaline solution 0.2 pg. of carbazole can be detected through the brilliant blue fluorescence of the anion. Carbazole can be characterized and estimated in the benzene extract of airborne particulates by the p-nitrosophenol spot test and spectrophotometric procedure, b y the 3-methyl-2benzothiazolone hydrazone (MBTH) spectrophotometric procedure, and by the excitation and emission spectra of the material in pentane, dimethylformamide, and alkaline dimethylformamide. The detection and determination of approximately 50 azo dyes and a smaller group of stilbene and Schiff base derivatives using 3methyl-2-benzothiazoloneare also described. of the 3 - methyl - 2benzothiazolone hydrazone (MBTH) spectrophotometric procedurr (14) to benzene extracts of some airborne particulates and to column chromatographed subfractions of these particulates (6) occasionally gave a brilliant blue solution, , ,A 586 mp. Consequently i t was desirable t o ascertain what types of organic compounds would react with the reagent. M B T H forms brilliant dyes with aromatic amines ( I d ) , imino heterocyclic c h pounds ( 1 4 , and formaldehyde ( I O ) . Preliminary results, which are being in-
vestigated, have indicated that azulene derivatives give a brilliant blue with the reagent. On the other hand, polycyclic hydrocarbons such as pyrene and perylene give negative results. I n the presence of an oxidizing agent, M B T H forms a strongly electrophilic diazonium salt. Compounds which are most subject to attack by electrophilic agents are characterized by a carbon atom which has a high electron density. Such a carbon atom is usually the negative terminal of a zwitterionic resonance system. On this basis, the mechanism for the determination of carbazole would be similar to that described for N,Ndimethylaniline (14). To characterize the compound (s) which gave a blue color in the M B T H procedue, numerous fluorescence and spectrophotometric methods were applied. Only those of value for carbazole will be described, e.g., the xanthydrol (2) and p-nitrosophenol procedures. The mechanism for the xanthydrol reaction with carbazole derivatives has been described ( I I ) . The mechanism for the p-nitrosophenol procedures probably consists of the following reactions:
benzene (p - phenylaaoaniline) derivatives are carcinogenic to animals (3). The ultraviolet absorption spectra of these amines have been reported (3). 4 - Aminoazobenzene compounds can be spectrally characterized by their basicity (1, 8, 12) and their Ca/Aa ratios ( b ) , Le., the relative proportion of the absorbances a t the long wave length maxima of the t n o tautomers (C and A) obtained in acid solution. However, in the trace analysis of a complev mixture for these compounds, analysis through the ultraviolet-visible absorption spectra of the pure compound would be immeasurably helped with the use of the more sensitive M B T H procedure (14) as an additional tool. The mechanism in the formation of the blue chromogen when hIBTH is reacted with N,N-dimethy1-4-aminoazobenzene (DAB) is believed to be similar to that described previously for parasubstituted N,N-dimethylanilines (14). Dimethylaniline reaction with 3methyl - 2 - benzothinzolone hydrazone by this procedure gives a chromogen . ,A 598 mp. E 83,000 (4, IS). Conwith sequently, the band a t approximately 600 mp obtained in the determination of
A
PPLICATION
1574
ANALYTICAL CHEMISTRY
Blue
Recent research has shown that many
4 - aminostilbene and 4 - aminoazc-
Green
DAB is believed to be derived from this chromogen by replacement of the
phenylazo group in the para position by the reagent. The band a t 672 m p is believed to be derived from the chromogen obtained b y substitution of the reagent in the ortho position to the dimethylamino group. I n t h e same way N-methyl-4-nminoazobenzene and N methylaniline, reacted with the reagent, should and do give a band a t approximately 580 m p while 4-an?inoazobenzene and aniline should and do give a band at approvimately 570 m p (Table I). In a previous paper ( I S ) spectral evidence was given for the postulate t h a t many para-substituted N,N-dialkylanilines are attacked by 3-methyl-2benzothiazolone hydrazone at the para position by replacement of t h a t particular group and/or at the ortho position. Attack of the reagent at the ortho position was postulated to account for the presence of bands a t approximately 635 and 670 mp. This latter type of band system can be seen in the absorption spectra obtained from N,N-dimethyl-4aminostilbene and N I X - dimethyl - pnitrosoaniline. I n the determination of S , N - dimethyl - 4 - aminobenzalnniline the spectrum showed bands a t 550, 625, and 670 mp, which indicated the presence of two chromogens formed from splitting of the molecule a t the CH=N band into aniline and N,h‘-dimethyl4-aminobenzaldehyde. The aniline was attacked by the reagent in the para position and gave the band near 580 mp. The original benzaldehyde derivative was attacked in the position ortho to the amino group. The bands at 625 and 670 mp were derived from this chromogen. All the Schiff bases reacted in this manner. The same bands were obtained a s were found in the separate fragments on reaction with the reagent. EXPERIMENTAL
Reagents and Equipment. MBTH hydrochloride (10) was prepared and purified by literature procedures and is also available from the Aldrich Chemical Co., Milwaukee 10, Wis. Dilute aqueous solutions of MBTH are stable for at least 1 week. Xanthydrol and p-nitrosophenol were obtained from Matheson, Coleman and Bell. The p nitrosophenol was recrystallized twice from xylene before use and the 0.5y0 solution of the compound in sulfuric acid was stable for about 4 hours. Acidic methanol contained 1 part concentrated hydrochloric acid and 5 parts methanol by volume. The azo dyes, stilbene derivatives, and other analogous compounds were prepared by standard literature procedures and crystallized from appropriate solvents to a constant melting point. A Cary Model 11 recording spectrophotometer with 1-cm. cells was used in the absorption spectral studies; the Aminco-Bowman spectrophotofluorometer with 1-cm. cells was used for the fluorescence studies; the American
Optical Co. rapid scan spectrophotometer was used in studying unstable colors. Spot Tests for Carbazole. pNITROSOPHENOL PROCEDURES. Test I. T h e test is conducted in t h e depression of a spot plate. To 1 drop of Table
1.
a 0.5% solution of p-nitrosophenol in sulfuric acid is added 1 drop of methanolic or acetic acid t e s t solution. I n t h e presence of carbazole a green color is obtained, identification limit, 0.4 pg. Then 1 drop of water is added. I n the presence of carbaaoIe the green
Spectrophotometric Determination of 4-Aminoazobenzene Derivatives and Azobenzene Analogs. L
Compound 4-Aminoazobenzene 4-Aminoazobenzene = AB N-Methyl AB
X
xm*x,
,
e
x
Compound mp Derivatives 4-Aminoazobenzene Derivatives 571 14 3’-Methyl DAB 603 68 644s 5 664s 52 589 36 3’-Nitro DAB 610 54 630 34 665s 43 664s 32 3‘-Trifluoromethyl DAB 608 69 3’-Methyl-N-methyl AB 586 36 663s 57 625s 34 4’-Acetyl DAB 606 25 664s 32 666s 20 N-Ethyl AB 605 67 4’-Amino DAB 585 41 666s 53 14 668s N-Phenyl AB 49 4’-Ethoxy DAB 623 39 598 4’-Methylthio-N-phenyl 624 9 665s 29 AB 4’-Ethyl DAB 603 59 N,N-Dimethylaminoazo- 604 70 662s 43 benzene = DABb 672 56 4’-Fluor0 DAB 603 37 2-Methyl DAB 588 72 664s 26 20 4’-Methyl DAB 664s 60 1 45 2’-Amino DAB 45 608 666s 31 4’-Sulf0 DAB 660s 57 612 37 2’-Chloro DAB 613 49 666s 73 64 664s 4’-Thiocyano DAB 599 18 2’-Ethyl DAB 67 12 603 668s 2,2’-Dimethyl DAB 665s 47 71 588 2’-Methoxy DAB 605 64 665s 15 665s 51 Azobenzene Analogs 2’-Methyl DAB 603 69 4Phenylazo-1-naphthyl- 587 674s 45 40 2-Methyl-2 ’-methoxyamine 583s 28 carbonyl DAB 665s 8 1-[N,N-Dimethyl-4608 66 2,3’-Dimethyl DAB 588 71 aminophenylazo]662s 55 670s naphthalene 14 2-Methyl-3’-chloro DSB 588 71 12 2- [N,N-Dimethyl-4 573 19 665s 4 aminophenylazol680s 2-Methyl-4’-acetyl DAB 587 65 fluorene 666s N,N-Dimethyl-4-amino- 632 13 116 2-hlethyl-4’-methylthio 585 74 stilbene0 119 667 D.4B 670s 10 2- [N,N-Dimethyl-4 630 28 2’,5’-Dimethyl DAB 601 66 aminophenyll-36658 26 665s phenylquinoxaline 43 2’,4’,6’-Tribromo DAB 612 671 5 N- [4-Hydroxybenzyli54 674s 4 dene] aniline 17 675 N-Methyl-N-ethylamino- 603 5s Ar- [N,N-Dimethyl-484 580 azobenzene = MEAB 663s aminobenzylidene] 32 71 625 2’-Chloro MEAB aniline 605 63 69 670 2-Nitro-9-[NJN-Di659s 51 628 18 2’-Nitro MEAB 42 methvl-4-amino-N613 17 667s 665s 34 benz$lidene] fluorene 3’-Acetamino MEAB 60.1 69 .. 2- [N,N-Dimethyl-4 21 634 amino-N-benzylidene] - 668 659s 21 33 3’-Nitro MEAB 54 605 1,3-indandione 660s 33 N ,N-Dimethyl-&p-di637 56 4’-Ethyl MEAB 600 60 cyano-4-vinylaniline 56 667 29 665s N[N,N-Dimethyl-4630 38 4’-Fluoro MEAB 601 64 amino-N-benz ylidene] - 670 38 663s 33 p-nitroaniline N-Methyl-N-benzyl -4B 607 10 2 [N-Benzylideneaminol- 631s 36 4 678s fluorene 667 39 N,N-Diethyl AB 570s 1[X,N-Dimethyl-432 90 637s 600 amino-A;-benzvlidene- 677 38 102 674s 5 aminolpyrene ” 49 755s 3’-Acetamino DAB 605 70 2 [p-Hydroxy-N-bena yli- 625s 24 664s 55 deneamino]fluorene 670 28 3’-Amino DAB 588 50 N,N-Dimethyl-4-amino- 633 48 25 672s benzaldehyde-2665 47 3’-Chloro DAB 608 66 quinolylhydrazone 665s 54 N,N-Dimethyl-4-amino555 37 3’-Ethoxy DAB 70 603 benzaldehyde-2-benzo- 620 34 663s 54 thiazolvl hydrazone 668s 31 ” All values based on minimum of two determinations and are within 1 2 y 0 of average. * Molar absorptivity values based on 36 determinations giving a value of 70,000 =t1000 a t Xmsx 604 mp. Value at 667 mp was 119,000 f 3000. mp
10x3
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1575
color turns blue. I n this last step the identification limit is about 2 pg. In both cases the blank is yellow. Test 11. The test is conducted on glass fiber filter paper (KO. 934-AH, H. Reeve Angel & Co., Clifton, N. J.). To 2 drops of 0.5% p-nitrosophenol in sulfuric acid is added 1 drop of methanolic test solution. After a 1-minute waiting period the color is noted. I n the presence of carbazole a bright bluegreen color is obtained. The identification limit for carbazole is 0.6 pg. The blank is yellow, N-Methyl- and N-ethyl-carbazole have identification limits of approsimately 1 pg. FLUORESCENCE PROCEDURE. To 1 drop of a solution of the sample in dimethylformamide on a filter paper is added 1 drop of 10% aqueous tetraethylammonium hydroxide. The fluorescence color under ultraviolet light is noted immediately, for it gradually fades. A brilliant blue fluorescence with a n identification limit of 0.2 pg. is obtained with carbazole. 9-Alkylcarbazoles show the same light blue fluorescence in dimethylformamide as in alkaline dimethylformamide, while carbazole has little if any fluorescence in neutral or acidic dimethylformamide solution. Spectrophotometric Procedures. NITROS SO PHENOL PROCEDURE. The solid sample, such as a n organic residue from airborne particulates, containing 10 to 300 pg. of a carbazole compound is dissolved by shaking with 1 ml. of a concentrated sulfuric acid solution containing 0.5% of p-nitrosophenol. Five minutes after the reagent has been added the mixture is diluted to 10 nil. with conrentrated sulfuric
Table II.
Compound Carbazole Carbazole
acid. The absorbance of the green solution is then read at the wave length maximum within 15 minutes after dilution to the mark. M B T H PROCEDURES: To 1 nil. of the methanolic test solution is added 1 ml. of aqueous RlBTH hydrochloride followed by 2 nil. of aqueous ferric chloride. The mixture is shaken, allowed to stand for the necessary time, then diluted to 10 ml. with the appropriate solvent. For the determination of carbazole 0.4% aqueous M B T H hydrochloride, 1.0% aqueous ferric chloride, a standing time of 30 minutes, and a diluting solvent of acidic methanol are used. The absorbance is determined at the wave length maximum of 586 mp against a blank. For the determination of 4-aminoazobenzenes, 4-aminostilbenes, Schiff bases, etc., 0.2% hydrochloride, 1.3% ferric chloride, a standing time of 15 min., and a diluting solvent of methanol are used. The absorption spectrum can be determined within 20 minutes for N , S dimethyl-4-aminoazobenzene and was determined within 5 minutes for the remainder of the test compounds. The wave length maxima and molar absorptivity values of a large number of compounds are given in Table I. RESULTS A N D DISCUSSIPN I
Absorption and Fluorescence Spectra of Carbazole. Probably the best means of characterization of carbazole (or its derivatives) is by its ultraviolet absorption spectra in a nonpolar solvent. The wave length maxima in millimicrons and molar
Excitation and Emission Spectra
Solvent DMF illk. DMF
Carbazole Pentane Carbazole fract. of Pentane air sample Benzene-soluble DMF fraction of air particulate matter Benzene-soluble Alk. DMF fraction of air particulate matter
Compound Concn.
Excit , A,
Emission Am,,; mp (
mP
x 1 0 - 7 ~ 298 347 ( 0 . 8 2 ) ; 360 (0.83) 1 x 1 0 - ~ M 292 425 (1.47); 450 (1.35): 475 (0.54) 2 x 10-6M 292 332 (1.53); 349 (1.68) 5
292 332; 349
11 pg./ml.
298 347s; 360; 408x; 434sx
11 pg./ml.
292 347x; 360x; 389x; 425; 450; 475s Emiss. A, my
Carbazole
DhlF
360
Carbazole
Alk. DMF
425
Carbazole
Pentane
349
Carbazole fract. of Pentane air sample 11 pg./ml. DMF Benzene-soluble fraction of air particulate matter 11 pg./ml. Benzene-soluble Alk. DMF fraction of air particulate matter MT values are microphotometer meter reading s = Shoulder. x = Unknown band. 0
1576
ANALYTICAL CHEMISTRY
349
Excitation; , ,A, mfi (MT) 298 (0.73); 330 (0.31); 342 10.29) 292 (1.32); 312 (0.54); 383 (0.24); 400 (0.21) 250 (1.41); 292 (2.7); 321 (0.93); 332 (0.87) 250; 292; 321; 332
360 298; 317x; 342 450 292; 312; 350sx; 364x; 38.1 times the meter multiplier reading.
absorpti:ity values in pentane are as follolls: 235 (-30,400), 246 (20,400), 255 (ll.SOO), 274s (5800), 281 (10,500), 285 (13.000), 291 (19,900), 307 (2800), 318 (3600), 323s (2600), and 331 (3800). There is much fine? structure and the bands are sharper than in the polar solvents. However, in a complex mixture, carbazole must be separated before attempting an absorption spectral study. The absorption spectra of carbazole in neutral and alkaline dimethl lformamide are additional help in detecting and determining carbazole after separation from a mixture,. The alkalization of a dimethylfornianiitle solution (0 1 nil. of 25% aqueoub tetlaethylammonium hydroxide per 10 ml. of DNF) of a carbazole compound containing a ring S H grouping causes a strong rrd shift in the long wave length band.. Formation of the carbazole anion also causes a threefold increase in thc ahsorbance of the 290-mp band, but the anion is unstable and measurements on the> alkaline solution must be made inimi~diately. Carbazole in dimethylformamide gave bands a t : 294 (16,000), 326 (3400), and 338 (3100) while carbaAole disso1vc.d in alkaline dimethyliormamide gax e bands a t 289 (44,000), 811 (9800)) 322s (5000), 370s (21,0@0), 382 (2600), and 402 (2200). These spectrophotometric procedurrs are much less qensitive and selective for carbazole than spectrophotofluorometric procedurrs. It is true of many fluorescent compounds that the excitation y m t i u m obtained a t a n appropiiate cniission spectral maw lrngth nia\inium is much more sensitive and much more Fclective than the absorption spectrum of the same niolecule 16, 7 , 9). The chief problems in spectrophotofluormictric analysis are the quenching effect of other compounds in the mixture and the greater difficulty in 011taining quantitative data on the spectrophotofluorometer as compared to a spectrophotometer. A dimethylformamide solution of carbazole shows a striking change in the rxcitation and emission spectra when the solution is made alkaline nith a strong base (Table 11). The excitation and eniiss:on spectra of carbazole in pentane (Table 11) are also extremely useful in the characterization and determination of carbazole. The excitation spectrum of carbazole in pentane or dimethyl formamide strongly resembles the abcorption spectrum in the same solvent. Thus, with a knowledge of organic absorption spectrophotometry, the more sensitive and highly selective excitation spectrum could be of value in the identification of an unknown fluorescent compound. The various procedures cover a nide range of sensitivity. By the vanthydrol method approvimately 80 pg of carba-
zoic. ~ x i i i i e rict,ectetl si)ccti.ol)lic)tometrically; by the ultraviolet absorption spectrum or the 3-mcthy1-2bciizothiazolone hydrazonc or p-iiitrosophenol procedures approxiniatcly 4 pg. of carbazole can be detectctl; by the excitation or emission spectrum in :i nonbasic solvent, 0.001 p g . of carbazolc can be detected spectrophotofluoromrtrically. The range of selectivity of thwP d i v w e methods will be discusscd. p-Nitrosophenol Procedure. Variables in t h e p-nitrosophenol procedure for the determination of carbazole n-ere investigated. Lower concrntrations of p-nitrosophenol gave lower in t en sities. -1t con c en t ra t,ion< of 0.5 t o 2.0cc of the reagent a plateau n ' a p reaclicd. Elon--.ver. the blank bccanie much more intcnsc, with progresFivrly hig1it.r concc~ntratioiisof thrs rcxgent. Consequently, a conccntwtioti of 0.5% of the nitrosophenol iii tlro migriit ~ v a z chosen ;is giving t,hc most rlt4r:ihlt. results ' I k color intensity was stablcs for Lit least 12 minutes after t h r solution had bwti diluted to 10 nil., then it grstlually tlecrensrd. Beer's law WIF oheyed from 4 to 100 p g . of mrbazolc. The nitrosophenol nwthod gavc LIPprosiiiiately tv-ice the molar absorptivity of t'lie santhydrol procedure (Figure l ) , but required iisc of concrntratPtl sul furi c n cid. Pol!-cyclic hydrocarboiis siicli :is pyrc'tir, :iuthracenr, and hpiizo [nlpyrene intrrfc~ed much l i w than cspccted, evcn though they form coloretl soliltions in roiiccntrated sulfuric acid. Phc~iiol. anisole. iY,.V - dimc~t,liylaniliiit~. pyrrolt., itidole, iiaphth:il~ne,1-iinphthol. 1 nictlios!.iiaphtlial
20 r,
500
600
xx)
X,mIJ
Figure 1. Visible absorption spectra obtained in determination of c a r b a zole(5 X 10-jM)
~ - : IIC i
h w c tn.0 procedures should bt> of i n c'li:iract,erizing carbazol(, rlriiyn-
on t,hc st~:ilitlurtl proccclure in the det,erminatioii of carbazole i> summarized here. When the percentage of methanol in the :ipproximately 4 ml. of reaction iiiixtur.e was doubled to the intensity decreased to ahoiit, 5OYc% 0.4 of the original value. Although the methanol interfered, the 1 ml. used in the standard reaction mixture n-as necessary to diaqolve the carbazole test sample. A concrntration of 0.27, of 3-methyl2-benzothiazolone hydrazone in the reagent gave an absorbance of 1.81 for a final carbazole concentration of 5 X lO-;Jf. Lovier concentrations gave Ion-er ~-alucs.while concentrations of 0.35 to 2.0Yc gave absorbance values of 2.00. A conctntration of 0.4% reagent wis chosen iwcause i t gave much lighter yellow blanks than were obt'aiiied with mow conctwtrnted reagent. The usc of 0 . 5 , 1.0. and 2.07, ferric chloridc gnvc absorbariccs of 1.41, 2.00, and 2.10, respecti\-ely. for a 4.5 X lO-'Jf find conepiitration of carbazole. Lon-er conccntrations of ferric chloride gave lon-er values: higher concentrations gave gweiiish blanks. The 1% ferric chloride solutiori gave a somedint lighter 131nnk t h i n the 2% solution. 'I'hr reaction time study resulted in a n nhsorhnncc time curve which did not rrach a plateau even after 1 hour. The increase in :ilisorbancc is sterp during the first fen- minutes but levels off . 30 minute.;. and tliereonly a slight increasc of 0.04 absorbance unit by 60 minutcls. For thcxse reasons :i 30-minute reaction
MBTH Procedures. For Carlinzole: T h e standard procedure !?-a' developed from a study of t h e manifold variables. For a better understanding, bhe effect of each \-ai%il)lc
After the 3-minute Ti-aiting period the addition of 0.2 to 1.5 nil. of concentrated hydrochloric acid follomd by methanol to 10 nil. gave optimum result's. With less than 0.2 ml. of hydrochloric acid the
in the proctdurc~. Ch1):izole gave LL ino1:ir absoi,ptiT.ity of 40,000 at, the !Val-e leiigtli tn:ixiniuii> of 6% m p . The 9-methyl and 9-csthj-1 r l c ~ i ~ t t i v of e s carhazolr (lid not wact as n-ell. but gave the same wave length maximum and a niolar absorptivity of 26.000. %.Acetyl carbazole reactcc? poorly :it first hut t,hc intensit.!- grndu d l y iiiereascd n-it11 hydrolysis of th(, coinpountl to carbazole. 1Ioilific:ltion of thc proretluro for carlinzolc by dilution n-ith n.otrr n-hilcb coolins. insttwl of dilution vit,li sulfurit. n&l. g : i w a blur solution of t h r mono1 'on. ,,A , 615 nip and E 12.000. i-r'i-cr. the color fa!lctl f : d y quickly :il)out 8 minutes the intensity rlc-
-
intcw-ity was diminishtld; n-ith more than 1.5ml. the test solutions containing carbazole became greener, although the nave length maximum and the intensity remained about the same. Volumes of acid less than 1.0 ml. tended to give turbid solutions. K i t h the use of the :iridic methanol solution (1 volume of concentrated hydrochloric acid to 5 volumes of mcthanol) as a diluent, optimum results n ere obtained. The color intensity n a s stable for 2 hours. I n 17 hours the absorbance dropped from an initial value of 2.00 to 1.83. Beer's law was obeyed from 4 to greater than 90 pg. of carbazole per 10 nil. of final solution. Reproducibility ~ a more q satisfactory than with the p nit1 osophenol procedure. Absorbance d u e s for the method agreed within 12%. The absorption spectra obtained in the santhydrol, p-nitrosophenol, and 3-methyl-2-benzothiazolone hydrazone determinations of carbazole are compared in Figure 1. Carlxizole gave a wave length maximum of 586 nip and a molar absorptivity of 44,000. 9-Methyl and 9-ethyl carbazole al-o reacted readily with the reagent giving bands at 595 and 597 mp, rcspcctively. Both derivatives gave a molar absorptivity of 49,000. Xnthanthrene, coronene, and benzo[g,h.i]perylene, hydrocarbons which could be present in a poorly separated carbazole chroniatographic fraction, gave ncgative results. Benzaldehyde also did not react. However. aliphatic aldehydes ( I O ) , aromatic amines ( I 4 ) , and imino heteroaromatic compounds (14) vi11 react. For example indole gave hands a t 462 and 510 mp with molar absorptiT-ities of 19,000 and 23,000, respectively. Ordinarily, aliphatic aldehydrs and the basic aromatic amines would not be fount1 in an airborne particulate fraction. The type of absorption spectrum obtaincrl with an aliphatic aldehyde is characterized by a doublet a t appro+ mately 635 and 6 i O mp and could not be mistaken for the spectrum obtained with carbazole. Foi DAB: T'ariahles in the standard procedurr for the drtermination of DAB were inveqtigated. Attempts to decrease the dilution factor (e.g., l ml. of test solution diluted t o 10 ml. of final volume) so as to increase the sensitivity resulted in turbid solutions. Optimum result5 were obtained a i t h 0 . l j to 0.2001, of the hydrazone reagent. I n the oxidation step maximum absorbance was obtained a t 1.2 to 1.4% ferric chloride solution. Before the final dilution, the reaction mixture contained approximately 25% methanol. If the percentage of methanol was doubled in this 4 ml. of mixture, the molar absorptivity mas halved. The amount of methanol could not be decreased because of solubility problems n i t h the
I
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VOL. 33,
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1577
test substance. The oxidation time was approximately 10 minutes for the attainment of maximum intensity. The color intensity was stable for approximately hour. From an initial value of 1.74, the absorbance dropped 0.01 unit every hour and a half so that after 19 hours of standing the absorbance was 1.62. If the final diluent mas concentrated hydrocliloric acid instead of methanol, the solution was completely decolorized. Beer’s law was obeyed from 1 to more than 70 pg. of DAB per 10 ml. of final solution. The molar absorptivity was 70,000 1000. The absorptivity obtained for DAB in the procedure was 0.31 pg.-I ml. cm.-’. One part of this dye could be detected in 360,000 parts of test solution. The spectral detection limit (absorbance = 0.1) for this dye in the standard spectrophotometric procedure was approximately 3 pg. This figure could be decreased by using proportionately smaller volumes. Determination of Azo Dyes. ,4pproximately 50 azo dyes were determined by the standard procedure developed for DAB. T o obtain optimum results for any other compound in Table I conditions would have to be varied to some extent. Most 4-amino, 4-alkylamino, and 4-dialkylamino azobenzenes gave intense blue to green colors in the procedure, Table I. Derivatives of 2-methyl-DAB gave very high intensities. The characteristic absorption spectrum obtained with this particular type of compound displayed a strong band of approximately 590 mp and a shoulder a t approximately 670 mp with one fourth to one seventh the intensity of the main band. In some cases either steric hindrance or substitution in the extended para position of a 4aminoazobenzene had an adverse effect on the determination of that particular compound. The following compounds gave negative results in the test: azobenzene, 4-hydroxyazobenzene, 4 methylmercaptoazobenzene, and 4‘ nitro - N,N - diethyl AB. The following compounds gave a weak reaction shown by a band a t approximately 630 or 670 mp with a molar absorptivity less than 4000: 4‘-nitroN - phenyl AB, 4’ - phenyl - DAB, 4’nitro - N - methyl - N - ethyl - AB, 3methyl-DAB, and N,N-dimethyl-p-(p2 - tolylazo - 2 - tolylazo) aniline. The compounds-4’-acetyl-DAB, 4’-methylmercapto - N - phenyl - AB, 2’,4’,6’tribromo - DAB, 4’ - thiocyano - DAB, and 2’ - methoxycarbonyl - 2 - methylDAB did not react completely for they showed the presence of a substantial amount of unreacted matenal. The nonreactivity of 3-methyl-DAB is in line with the very weak reaction of N,Wdimethyl-o-toluidine with the reagent. Determination of Azo Dye Analogs. Approximately 25 compounds of this type have been reacted (Table I).
*
-
1578
ANALYTICAL CHEMISTRY
Only a few stilbene-type derivatives were available for investigation. Of these compounds, N,N-dimethyl-4aminostilbene gave an extremely intense reaction with a molar absorptivity of 119,000 and an absorptivity of 0.53 pg.-‘ ml. cm.-’ As sensitive methods for the trace analysis of aminostilbene compounds are not available in the literature, this procedure should fill that need. Various types of Schiff bases gave positive results in the procedure (Table I). Consequently, the methods could be used for the detection, characterization, and determination of Schiff bases. Xegative results were obtained for the following compounds-1,l-dimethylhydrazine, 2 - methylaminoethanol, malonic acid, hexamet hylenetetramine, N - methyl - 2 - pyrrolidone, 8bromostyrene, piperonylic acid, 1methyl - 1 - phenyl - hydrazine, N,K’methylenebisacrylamide, 2 - methylmercaptobenzothiazole, benzaldehyde, p - hydroxybenzaldehyde, and 4 hydroxybiphenyl. 3 - Methyl - 2 - benzothiazolone hydrazone has been used for the determination of aliphatic aldehydes, aromatic amines, polycyclic imino heteroaromatic compounds, and azulene derivatives, so that any of these types of compounds could interfere in the determination of azo, stilbene, or Schiff base compounds. Another family of compounds that reacted readily with the reagent were the pyrrole derivatives. ’With pyrrole, wave length maxima of 600 and 664 mp and molar absorptivities of 16,000 and 34,000, respectively, were obtained. NMethylpyrrole gave bands a t 612 and 673 mp with molar absorptivities of 37,000 and 49,000, respectively, while 2,5-dimethylpyrrole gave equally intense bands at 635 and 657 mp with a molar absorptivity of 28,000. Because of the general utility of the reagent, care must be exercised in the detection or determination of any particular compound or group of compounds that other interfering chemicals are not present, can be masked, or can be separated from the entity being determined. APPLICATION
The evidence for the characterization of carbazole in the approximately half dozen airborne particulate samples in which it was found is summarized here. Column chromatography on alumina of a mixture of known polycyclic hydrocarbons by a standardized procedure (6) clearly indicates that carbazole comes through the column directly after coronene. A similar chromatographic procedure was utilized for several benzeneextracted fractions of airborne particulates that gave a blue color, A,., 586 mp, in the 3-methyl-2-benzothiazolone
hydrazone procedure. In these air pollution samples the fraction follwing coronene had an ultraviolet absorption spectrum practically identical to that of carbazole. One characteristic of the spectrum of this fraction must be mentioned, and that is the presence of sharp, well-defined, but much less intense bands a t 346, 364, and 383 mp, The structure of the compound($ from which these bands are derived is unknown. The excitation and emission spectra in pentane of the carbazole fractions and pure carbazole were identical (Table 11). The excitation and emission spectra of the carbazole fractions in acidic or alkaline dimethylformamide were also identical to the analogous spectra of carbazole. The compound found in these air pollution samples is, without a doubt, carbazole. Comparison of the 3-methyl-2-benzothiazolone hydrazone, xanthydrol, and p-nitrosophenol procedures for the determination of carbazole in airborne particulates indicates that the p-nitrosophenol method is the simplest. The 3-methyl-2-benzothiazolone hydrazone and the p-nitrosophenol procedures are the most sensitive for the determination of carbazole. A test sample mould have to contain 20 times as much carbazole for the xanthydrol method (2) to give the same intensity as obtained with the 3 - methyl - 2 - benzothiazolone hydrazone procedure. However, the xanthydrol method should be capable of a further improvement in sensitivity by slight modification. Thus far, no evidence has been found for any other compound present in airborne particulates reacting with the hydrazone reagent, so the method appears to be specific for carbazole in airborne particulates. -4 carbazole fraction obtained from an airborne particulate sample was quantitatively analyzed by the various methods (Table 111). The methods averaged 67 3 pg. of carbazole. The application of these spectral procedures to the qualitative analysis of the benzene extracts of appropriate airborne particulates mas next investigated. The fluorescence spot test was of no value here. The p-nitrosophenol spot plate test worked very nicely and a bright green color was obtained. The detection limit for carbazole in benzene extracts of airborne particulates was about 1 pg. The air samples containing carbazole were analyzed by the 3-methyl-2benzothiazolone hydrazone and the pnitrosophenol spectrophotometrie procedures and gave spectra containing bands a t 586 and 695 mp, respectively. Consequently, duplicate recovery experiments were performed on a carbazole-free benzene extract of airborne particulates to which known amounts of carbazole were added (Table IV).
*
Table 111. Amount of Carbazole in Carbazole Air Fraction
Carbazole, Method 3-Methyl-2-benzothiazolone hydrazone p-Nitrosophenol UV spectra in pentane, total absorbance a t 291 mp UV spectra in pentane, base line method at 291 mg Emission spectra in pentane at X 349 mp - Excit. X 292 mp Excitation spectrum in pentane at, X 292 mp Emiss. X 349 nw
-
a. 65 67 71 66 67 64
When fairly large amounts of sample (1 to 2 mg.) containing 7 to 90 pg. of carbazole are t o be analyzed, any of the analytical procedures described in this paper can be used. However, in the 3-methyl-2-benzothiazolone procedure a slight turbidity may result, so that i t is necessary to run an extra blank containing the benzene extract and everything else except the hydrazone reagent. This extra blank corrects for the turbidity and more reasonable results are obtained. In the fluorescence methods very much lower concentrations of carbazole can be determined, but the quenching effect of the many other compounds in the mixture must be standardized by running a Beer's law study on carbazole solutions containing a standard weight
of a benaene air particulate fraction and analyzing for carbazole in this weight of material ( 7 ) . The excitation and emission spectra of a benzene fraction of a n air particulate sample at the appropriate wave lengths for the analysis for carbazole are compared to the analogous spectra of pure- carbazole (Table 11). Occasionally, a closer similarity of the corresponding spectra of the pollutants fraction and of carbazole has been obtained. Obviously, the methods show promise of further development. The type of air pollution associated with the presence of carbazole will be discussed in a separate publication, as will the atmospheric composition in terms of polycyclic compounds.
Table IV. Recovery of Carbazole from Air Samples
Carbazole. Found Anal. a t Emiss. p-Ni- X 348 Hydra- troso- Exzone phenol cited meth- meth- at X In 292 od sample od
69
68
55
...
LIE.
.
I
Anal. a t Excit. X 292 Emission at X 348
...
(9) Sawicki, E., Hauser, T. R., Stanley, T. W., Int. J . Air Pollution 2, 253 (1960). (IO) Sawicki, E., Hauser, T. R., Stanley, T . W., Elbert, W.,ANAL.CHEW 33, 93 (1961). (11) Sawicki, E., Oliverio, V. T., J . Org. Chem. 21 , 183 (1956). (12) Sawicki, E., Ray, F. E., Ibid., 19, 1686 (1954). (13) Sawicki, E., Stanley, T. W., Hauser, T. R., Barry, R., ANAL.CHEM.31, 1664 (1959). (14) Sawicki, E., Stanley, T. W., Hauser, T. R., Noe, J. L., Ibid., 33,722 (1961). RECEIVED for review January 30, 1961. Accepted July 13, 1961. Work performed a t the Laboratory of Engineering and Physical Sciences, Division of Air Pollution, Public Health Service, U. S. Department of Health, Education, and Welfare. Divisions of Analytical and Water and Waste Chemistry, 140th Meeting, ACS, Chicago, Ill., September 1961.
LITERATURE CITED
(1) Badger, G. M., Buttery, R. G., Lewis, G. E.. J . Chem. SOC.(London) 1954, '1888. ' (2) Gilbert, G., Stickel, R. M., Morgan, H. H., Jr., ANAL. CHEM. 31, 1981 (1959). (3) Haddow, A., Harris, R. J. C., Kon, G. A. R., Roe, E. M. F., Phil. Trans. Roy. SOC.London Ser. A 24, 147 (1948). (4) Hunig, S.,Nother, H., Ann. 628, 69, 84 (1959). (5) Sawcki, E., J . Org. Chem. 22, 621 (1957). (6) Sawicki, E., Elbert, W., Stanley, T. W., Hauser, T. R., Fox, F. T., AKAL. CHEM.32,810 (1960). (7) Sawicki, E., Elbert, W., Stanley, T. W., Hauser, T. R., Fox, F. T., Int. J . Air Pollution 2,273 (1960). (8) Sawicki, E., Gerber, D., J . Org.Chem. 21,410 (1956).
Dete r min at io n of Excita ti o n S pect ra with a Recording Spectrophotometer CLAUDE W. SILL Health and Safety Division, U . S. Atomic Energy Commission, Idaho Falls, Idaho
b A simple and inexpensive accessory has been developed to permit the determination of excitation spectra of fluorescent materials with a recording spectrophotometer. The accessory consists of three front-surfaced mirrors arranged so that the light from the monochromator i s redirected to pass through a sample cell at a right angle to the direction taken b y the fluorescent light from the cell to the phototube compartment. The signal from the detector reflects the change in intensity of fluorescent light as a function of the wave length of the light incident upon the sample. Resolution and stray light characteristics o f the resulting spectra are as good as those of the spectrophotometer used. After cor-
recting for the emission characteristics of the light source, excitation spectra o f fluorescent materials parallel closely their absorption spectra and can be used in a similar manner as an analytical tool. However, the excitation spectra may be thousands of times more sensitive and applicable in the presence of other absorbing but nonfluorescent species that would interfere seriously with absorption measurements. Several examples of practical importance are given.
M
EASUREMENT
OF
FLUORESCENCE
has long been used for quantitative determination of many organic compounds of biological interest such
as vitamins, alkaloids, etc. Because of their high inherent sensitivity and selectivity, fluorometric methods are now being investigated with increasing frequency for the determination of microgram and submicrogram quantities of metals. Although considerable attention is generally directed toward choosing the proper filter for transmitting the fluorescent radiation to the detector, relatively less effort is expended in determining the most advantageous wave lengths with which to produce the fluorescence. This unfortunate situation has arisen and is still present mainly because of lack of instrumentation for determination of the necessary excitation spectra and/or consequent lack of information conVOL 33,
NO. 11, OCTOBER 1961
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