Nonspecifkity of the Gibbs Reaction Jack C. Dacrel Toxicology Research Unit, Medical School, Unisersity of Otago, Dunedin, New Zealand
THEREACTION between phenols unsubstituted in the para position and 2,6-dibromo- or 2,6-dichloro-p-benzoquinone4-chlorimine (Gibbs reagent, more commonly called 2,6dibromo- or 2,6-dichloroquinone-chloroimideand less commonly N,2,6-trichloro-p-quinoneimine) is usually called the Gibbs Reaction (I-@. Numerous phenols substituted in the para position, however, have been reported t o give a positive Gibbs reaction (7-23). Some of these and many other para substituted phenols, especially those used as anti-oxidants, were submitted to the Gibbs reaction; the colored indophenols formed were examined spectrometrically and the results are reported in this paper. EXPERIMENTAL All measurements were carried out using a Model D K 2 recording spectrophotometer (Beckman Instruments Inc., Fullerton, Calif.). Compounds. All the solvents used were of “Analar” quality. The 2,6-dichloro-p-benzoquinone-4-chlorimine was purchased from Hopkin and Williams Ltd., Chadwell Heath, Essex, England, and was recrystallized to constant mp. All other compounds were recrystallized and checked for purity by paper chromatography. Present address, Laboratory of Environmental Medicine, Tulane University School of Medicine, 1700 Perdido Street, New Orleans, La. 70112 (1) H. D. Gibbs. Chem. Rec., 3, 291 (1927). (2) H. D. Gibbs, J . Biol. Chem., 71, 445 (1927). (3) Zbid., 72, 649 (1927). (4) H. D. Gibbs,J. Phys. Cliem., 31,1053 (1927). (5) H. D. Gibbs, W. L. Hall, and W. M. Clark, Supplement No. 69 to the Public Health Reports, U. S. Public Health Service, pp 1-35 (1928). (6) J. R. Baylis, J . Amer. Water Works Ass., 19, 597 (1928). (7) V. E. Davidson, J. Keane, and T. J. Nolan, Sci. Proc. Roy. Diibliti Soc., 23, 143 (1943). (8) T. J. Nolan, J. Algar, E. P. McCann, W. A. Manahan, and N. Nolan, ibid., 24, 319 (1948). (9) A. W. Beshgetoor, L. M. Greene, and V. A. Stenger, IND. ENG. CHEM., ANAL.ED., 16, 694 (1944). (10) C. T. Calam, P. W. Clutterbuck, A. E. Oxford, and H. Raistrick, Biocliem. J., 41, 458 (1947). (11) D. H. R. Barton and A. I. Scott, J . Chem. SOC..1958, 1767. (12) J. H. Birkinshaw, A. Bracken, E. N. Morgan, and H. Raistrick, Biocliem. J., 43,216 (1948). (13) G. F. Edwards, J . Ass. Ofic. Agric. Chem., 33, 855 (1950). (14) J. H. Mahon and R. A. Chapman, ANAL.CHEM.,23, 1120 (1951). (15) J. H. Birkinshaw, H. Raistrick, D. J. Ross, and C. E. Stickings, Biocliem. J., 50, 610 (1952). (16) W. M. Azouz, D. V. Parke, and R. T. Williams, ibid.,59,410 (1955). (17) W. R. Jondorf, D. V. Parke, and R. T. Williams, ibid., 61, 512 ( 1955). (18) J. C. Dacre. F. A. Denz, and T. H. Kennedy, ibid., 64, 777 ( 1956). (19) S. Kuramoto, R. Jenness, and S. T. Coulter, J. Duiry Sci., 40, 187 (1957). (20) J. H. Birkinshaw, P. Chaplen, and R. Lahoz-Oliver, Biochem. J . , 67, 155 (1957). (21) W. R. Jondorf. D. V. Parke, and R. T. Williams, ibid.. 69, 181 (1958). (22) D. V. Parke and R. T. Williams, !bid., 74, 5 (1960). (23) J. C. Dacre, ibid., 78, 758 (1961).
3,5-Di-tert-butyl-4-hydroxybenzaldehydewas prepared by bromine oxidation of 2,6-di-tert-butyl-4-methylphenol in tert-butanol and 3,5-di-tert-butyl-4-hydroxybenzylalcohol from 2,6-di-tert-butyl-4-bromo-4-methyl-2,5-cyclohexadienone as described by Coppinger and Campbell ( 2 4 ) ; 3,5-di-tertbutyl-4-hydroxybenzoic acid was prepared by oxidation of the aldehyde as described by Yohe etal. (25). The substituted 4-alkoxy-phenols were kindly given by Dr. J. A. Chenicek, Universal Oil Products Company, Riverside, Ill. 60546. Reagents. A solution of 2,6-dichloro-p-benzoquinone-4chlorimine was freshly prepared as a 0.1 (wt/v) solution in ethanol. The buffer was a 2.075 solution of sodium borate of p H 9.24, which was constantly checked. Qualitative Procedure. The substance to be tested (ca. 1 mg) was dissolved in ethanol (5 ml) and Gibbs reagent (5 ml) added. The mixture was diluted with a n equal volume of sodium borate buffer and well mixed. Those para substituted phenols giving a positive Gibbs reaction were then examined quantitatively. Quantitative Procedure. The compound (1 mg) was dissolved in ethanol (5 ml) in a calibrated flask a n d Gibbs reagent (5 ml) then added; buffer was added to bring the volume up to either 50 o r 100 ml and the solution was well mixed. This latter concentration usually proved satisfactory for most of the spectral measurements. Any necessary dilutions were made using the aqueous borate buffer. The blank was a precisely similar solution of the Gibbs reagent only in the ethanol/borate mixture. Measurements were made 20-30 minutes after mixing when the indophenol band, assuming complete reaction, had obtained its maximum. The 1butanol extracts were prepared by shaking out a n aliquot (usually 10 ml) of the colored indophenol in the borate buffer with a n equal volume of the solvent (26). The blank was similarly treated. RESULTS AND DISCUSSION p-Alkoxyphenols. Sixteen substituted 4-alkoxyphenols were studied and all reacted with the Gibbs reagent to produce the characteristic blue color a t p H 9.24 (Table I). Those phenols substituted in the 2- and 6-positions (2,6-di-tertbutyl- and 2-tert-butyl-6-methyl-4-methoxyphenol;see also Table 11) tended to give a typical magenta color (absorption region 565-575 nm). I t should be noted also that those phenols substituted in the 3- (or meta-) position (Table I) all have the molecular extinction coefficient below 10,000 in the buffer solution. Extraction of the indophenol color into 1-butanol increases the wavelength of the absorption peak in all cases (an average shift of 34 nm) and markedly increases the molecular extinction coefficient (average increase 4,310). Besides the p-methoxy group, the p-ethoxy and p-propoxy phenols also give a positive Gibbs reaction. It would appear probable that all phenols with a para-substituted alkoxy group can be regarded as being true exceptions to the Gibbs reaction.
(24) G. M. Coppinger and T. W. Campbell, J . Anier. Chem. SOC., 75,734 (1953). (25) G. R. Yohe, J. E. Dunbar, R. L. Pedrotti, F. M. Scheidt, F. G. H. Lee, and E. C . Smith, J . Org. Cliewz.. 21, 1289 (1956). (26) M. B. Ettinger and C. C. Ruchhoft, ANAL.CHEM.,20, 1191 (1948). ANALYTICAL CHEMISTRY, VOL. 43, NO. 4, APRIL 1971
589
Absorption Data for Phenol and Some 4-Alkoxyphenols after Reaction with Gibbs Reagent at pH 9.24 Ethanoljborate buffer 1-Butanol extract Molecular Molecular extinction extinction Wavelength, coefficient, Wavelength, coefficient, Phenol nrn E rnax nrn E rnax Phenol (26p 610 13,910 650 21,430 4-Methoxyphenol 615 14,380 655 19,000 2-Methyl-4-methoxyphenol 595 14,490 635 21,110 3-Methyl-4-rnethoxyphenol 610 9,980 650 17,660 2,5-Dirnethyl-4-rnethoxyphenol 595 7,900 625 8,660 2,6-Dimethyl-4-methoxyphenolb 580 12,770 615 14,740 2-12-Butyl-4-rnethoxyphenol 595 18,810 630 21,060 600 17,460 2-iso-Butyl-4-methoxyphenol 635 25,560 590 15,480 2-sec-Butyl-4-methoxyphenol 635 23,400 580 14,220 2-terf-Butyl-4-methoxyphenol (14) 620 20,340 3-rert-Butyl-4-rnethoxyphenol (18) 625 4,770 665 6,030 2,5-Di-terf-butyl-4-rnethoxyphenol 595 8,500 610 11,680 2,6-Di-tert-butyl-4-meth~xyphenol~ 565 15,100 585 18,460 575 13,000 2-rert-B~rtyl-6-methyl-4-methoxyphenol~ 605 16,880 610 7,660 3-tert-Butyl-6-methyl-4-rnethoxyphenol 630 7,950 580 13,200 2-tert-Butyl-4-ethoxyphenol 620 19,400 5 80 12,480 2-tert-Butyl-4-iso-propoxyphenol 620 17,260 a See references. b Magenta in ethanol; greenish blue in 1-butanol. Table I.
Table 11. Color and Absorption Data of Some Para Substituted Phenols after Reaction with Gibbs Reagent a t p H 9.24 Ethanoljborate buffer
Phenol Vanillyl alcohol ( I S ) 3,5-Di-tert-butyl-4-hydroxybenzyl alcohol (23) 3,5-Di-ferf-butyl-4hydroxybenzaldehyde (23) p-Hydroxybenzoic acid (7) Vanillic acid (19) 3,5-Di-fert-butyl-4hydroxybenzoic acid (23) Cyclopolic acid (15) Cyclopaldic acid (15) rior-Mycophenolic acid (12) Quadrilineation (20) p-Chlorophenol (7) p-Bromophenol (9) p-Iodophenol 2-Chloro-5-hydroxy toluene 4-Chlor0- 3 5-xylen-1-01 p-Chlorothymol
Molecular Molecular Waveextinction Waveextinction length, coefficient length, coefficient, Color nrn E rnax Color nrn E max Pura-Hydroxy substituted benzaldehydes, benzyl alcohols, and benzoic acids Blue 595 25,870 Greenish blue 635 36,340 Magenta Weak magenta Blue Blue
565
23,600
Blue
590
32,100
565 615 595
3,630 2,350 12,770
Blue Greenish blue Greenish blue
585 655 635
3,980 2,620 17,140
Magenta Blue Green-blue Magenta" Blue
565 660 680 595b 625
17,000 12,840 6,490
Blue Greenish blue Greenish blue Greenish blue Greenish blue
590 680 695 680 675
22,500 16,080 9,280
608 608
Para-Halogenophenols 10,670 Greenish blue 14,360 Greenish blue Greenish blue
652 652
19,670 26,470
605 630
16,680 9,400
Greenish blue Greenish blue
645 680
29,940 21,610
595 665 660
10,710 8,470 2,800
Blue Greenish blue Blue
620 685 680
18,470 9,820 4,990
Blue Blue Green-blue
Blue Blue Purplish blue Dihydroerdin ( I O , I I ) Blue Dihydrogeodin ( I O , 11) Magenta a Initially blue-green which quickly changes. Inflection at 510 nrn. ~
1-Butanol extract
p-Hgdroxybenzaldehydes, Benzoic Acids, and Benzyl Alcohols. Little is known of the reaction between the Gibbs reagent and phenols with one of the groups, alcohol, aldehyde, or acid, in the pura-position. All the compounds restudied in this group that gave a positive Gibbs reaction are listed in Table I1 together with their characteristic color, absorption maximum, and extinction coefficients. 590 * ANALYTICAL CHEMISTRY, VOL. 43, NO. 4, APRIL 1971
2,300
2,800
p-Halogenophenols. T h e irregular nature of the Gibbs reaction with p-halogenophenols was first shown by Beshgetoor, Greene, and Stenger (9). Other chlorophenols have been investigated by Williams and coworkers (16, 17,21). Some para substituted phenols that gave a negative Gibbs reaction are listed in Table 111. The Gibbs reaction has been used by many chemists, par-
studying the metabolism of phenolic compounds in experimental animals [see e.g. Bray and Thorpe (30)]a n d especially for their identification using paper chromatographic techniques [ e . g . Bray, Thorpe, and White ( 3 1 ) ;McIsaac and Williams (32); Mead, Smith, and Williams (33)]. The need for caution in the interpretation of the result of the Gibbs color reaction should now be clear; even more so in view of the additional facts that many highly substituted phenols with the para position unsubstituted d o not give a positive test [see e.g. Birkinshaw, Bracken, Morgan, and Raistrick ( 1 2 ) ; Briggs a n d Locker (29)], while many compounds, e.g. amines (Castle, 34) and other amino derivatives (Fearon, 35) also give purple, violet, or other characteristic colors.
Table 111. Some Para Substituted Phenols Giving a Negative Gibbs Reaction p-Halogenophenols 2,4-Dibromophenol 2,4,6-TrichlorophenoI 2,4,6-Tribromophenol 2,4,6-Triiodophenol 2-Hydroxy-5-chlorobenzaldehyde p-Hydroxybenzaldehydes P-Resorcyl-aldehyde 3-Ethoxy-4-hydroxybenzaldehyde p-Hydroxybenzyl alcohol p-Benzyl-phenol p-Hydroxybenzoic acids Protocatechuic acid P-Resorcylic acid Gallic acid
RECEIVED for review August 31, 1970. Accepted December 14, 1970. (30) H. G. Bray and W. V. Thorpe, in “Methods of Biochemical Analysis,” D. Glick, Ed., Interscience Publishers, New York, N. Y . ,Vol. 1, p 27 (1954). (31) H. G. Bray, W. V. Thorpe, and K. White, Biochenz. J.,46, 271 ( 1950). (32) W. M. McIsaac and R. T. Williams, ibid., 66, 369 (1957). (33) J. A. R. Mead, J. N. Smith, and R. T.Williams, ibid., 68, 61 ( 1958). (34) R. Castle, Chem. I d . (Lorzdorz),313 (1950). (35) W. R. Fearon, Biochem. J., 38, 399 (1944).
ticularly those working o n the elucidation of the structure of naturally occurring organic compounds [see e.g. King, King, and Manning (27); Hems and Todd (?8); Briggs and Locker (29)]. The reaction has also been applied both by those
_
~
_
(27) F. E. King, T. J. King, and L. C . Manning, J. Chem. Soc., 1957,563. (28) B. A. Hems and A. R. Todd, ibid., 1940, 1208. (29) L. H. BriggsandR. H. Locker, ibid.,1951,3131.
Rapid Hydrocarbon-Type Analysis of Gasoline by Dual Column Gas Chromatography R. E. Robinson, R . H. Coe, and M. J. O’Neal Shell Oil Company, Houston Research Laboratory, Deer Park, Texas 77536 AN OFTEN REQUIRED characterization of gasoline-range hydrocarbon streams is type composition in terms of total saturates, olefins, and aromatics. This analysis is conventionally carried out by the fluorescent indicator adsorption (FIA) method (1). T h e technique lacks good precision and becomes extremely difficult to apply to highly colored samples derived from pyrolysis, coking, hydrocracking, etc. Moreover, the procedure is rather lengthy. F o r light gasoline fractions, the analysis time is increased further because the sample must be depentanized first by distillation, with subsequent analysis of the Cs-and-lighter fraction by gas chromatography a n d finally analysis of the Cs- fraction by the F I A method. A more rapid and direct approach is t o employ a gas chromatographic method, wherein the entire untreated sample is injected into the chromatograph. Martin ( 2 ) successfully utilized a combined method for the separation of gasoline into aromatics, olefins, and total saturates, based o n group chromatographic separation and chemical absorption. Separation of aromatics from saturates a n d olefins was obtained with a @,@’-thiodipropionitrile packed column; olefins were then separated from saturates by reaction with mercuric perchlorate. This technique was (1) Am. SOC.Testing Materials, “1968 Book of ASTM Standards,”
Method D-1319-66T,Part 17, p 506. ( 2 ) R. L. Martin, ANAL.CHEM., 34, 896 (1962).
later extended to include n-paraffin separation from other saturates and individual n-paraffin analysis ( 3 , 4). M o r e recently, a subtractive method for the rapid analysis of hydrocarbon types has been described by Soulages ( 5 ) . The aromatic and olefinic hydrocarbons are selectively retained by two chemical absorbers in parallel while the total saturates pass unaltered, and the resulting peaks are detected by a single flame ionization detector. In the present paper, a new, but related, technique for rapidly determining hydrocarbon-type content of gasoline is described. A parallel capillary column system a n d a n olefinabsorbing trap are employed with valving. One column is uncoated and the other is coated with the highly polar N , N bis(2-cyanoethy1)formamide (6). This coated column is in series with the trap. By proper adjustment of flow rates, each column receives the same total sample from a splitter. The empty column passes its charge directly to the detector where a measure of total sample is obtained. The other column retards the aromatics and the trap removes the olefins
(3) D. K. Albert, ANAL.CHEM.,35, 1918 (1963). (4) L. E. Green, D. K. Albert, and H. H. Barber, J. Gas Chromatogr., 4, 319 (1966). (5) N. L. Soulages and A. M. Brieva, ANAL.CHEM.,in press. (6) M. Rogozinski and I. Kaufman, J . Gas Chromatogr., 4, 413 (1966).
ANALYTICAL CHEMISTRY, VOL. 43, NO. 4, APRIL 1971
591
