ON THE THEORY OF FLUORESCENCE’ BY GERTRUD WOKER
i
It is known that fluorescence and color may occur simultaneously in one and the same substance. This is usually the case when the following conditions are fulfilled: (a)Absorption bands in the ultra-violet or in the visible part of the spectrum. ( b ) Absorption bands in any part of the visible spectrum. (c) Reflection of, or transparency to, part of the visible spectrum. . ( d ) Change of a part of the absorbed rays into those of the visible spectrum. (e) Change of a part of the absorbed rays into heat. The conditions (a)and ( d ) are essential t o fluorescence and the others, ( b ) , (c) and (e) to color. Even when all these are satisfied, it may happen however that a substance, though colored, does not fluoresce. An actual, fluorescence may be masked by being identical with the color of the substance, as was pointed out by Richard Meyer’; it is also possible that an intense fluorescence may become invisible if it is complementary to the strong color of the substance itself or to a highly colored impurity. Thus an intense yellowish-orange color of the substance itself or of an impurity contained in it would nullify a blue or violet fluorescence. I have actually observed that the bluish fluorescence of quinine sulphate disappears on adding a solution of picric acid and that methyl orange masks the fluorescence of petroleum, On dissolving more and more picric acid in petroleum the fluorescence becomes weaker and weaker, finally disappearing. The naphthoflavanone, recently prepared by me, shows a beautiful bluish fluorescence only when the perfectly pure Translated from the author’s German niatiuscript by W, D. Bancroft. Zeit. phys. Chem., 24, 479 (1897).
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product is dissolved in colorless ethyl alcohol. If the alcoholic solution is colored yellow by traces of chalkone, the fluorescence cannot be detected. Over and above these observations, the following known facts illustrate the damaging effect of complementary colors. The violet fluorescence of pure chrysene is entirely masked by the yellow color of the impurity which it is so difficult to remove. The yellow solution of anthracene in sulphuric acid shows no trace of the blue anthracene fluorescence. The 3,2-diethoxyflavone1 dissolves in sulphuric acid at first with a yellow color. After standing awhile the solution becomes colorless. As the color disappears the clear blue fluorescence becomes visible and is a t its best when so much sulphuric acid is added that the solution becomes colorless. In the xanthone group, which has been studied carefully in the laboratory of Berne University by v. Kostanecki and his students, the violet and blue fluorescence is visible only when the solution is colorless and disappears when the solution is even faintly yellow2. By adding a solution with a color complementary to that part of the substance in question it ought to be possible to develop fluorescence in a non-fluorescing derivative of a fluorescing, colored substance, or to increase an actual fluorescence. In the most favorable case the existence of color will have no effect on the fluorescence, while in the most unfavorable case it will obliterate the fluorescence. This last case is very conspicuous in the anthracene group where the color and the fluorescence each3kills the other. Between these two extremes are to be found all possible intermediate forms in which the fluorescence is more or less affected or displaced by the color. Just for this reason in many cases, the fluorescence of a compound will be much damaged by the presence of chromophoric groups, especially of complex chromophores v . Kostanecki and v. Salis: Ber. chem. Ges. Berlin, 32, 1020 ( ~ S g g ) . The same thing can also be noticed in sulphuric acid solution. Liebermann : Ber. chem. Ges., Berlin, 13, 913 (18So). R . M e j e r : Zeit. phys. Chem., 24, 468 (1897).
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with intense colors. This can be seen most clearly by comparing the fluorescence of colored substances with those of the corresponding leuco-compounds. Liebermannl has shown that neither anthraquinone nor its derivatives are fluorescent, while fluorescence is to be found in pure anthracene compounds in which the ketone group has been reduced. The fluorescence of the anthraquinones is checked by the intensely colored complex chromophore 0 : C . C . C : 0. If this is more or less completely destroyed by the addition of hydrogen, the fluorescence phenomena of the anthracenes, anthranols and oxyanthranols appear, as described by I3ebermann.l As a further example we may cite the diphenylquinoxaline with the complex chromophore2 N :C. C : N . There is no fluorescence to this substance but there is to its leuco-compound, diphenyl-dihydroquinoxaline, in which one of the double bonds has been broken. Byaddition of phenol the diphenylquinoxaline can be converted into a fluorescing azonium base3 which is probably due to the fact that the introduction of another phenyl group develops new ultra-violet absorption bands in the compound and to these is due the increased fluorescence. The nitro group seems to be the most effective of all the chromophores in checking fluorescence. It is quite possible that this peculiarity is connected with the fact that most nitro compounds are yellow. From the facts previously cited it appears that a yellow color in alkaline solution causes the violet and blue fluorescence of the xanthone compounds to disappear and that the bluish fluorescence of petroleum and of quinine sulphate solution can be nullified by addition of methyl orange or picric acid. The complementary nature of the two sets of colors is apparently the cause of the phenomenon.
’ 1. c. 0. Fischer : Rer. chern. Ges., Berlin, 2 5 , 2826 (1892) ; 27, 719 (1894). 0. N. Witt: Ibid., 2 0 , 1183 (1887). R. Meyer: Zeit. phys. Chem., 24, 482 (1897).
*
Since the blue to violet fluorescence is the most common type, it is quite conceivable that yellow is the color which is chiefly antagonistic to fluorescence. Among chromophores the most antagonistic would be those causing a yellow color, and therefore pre-eminently the nitro group. The facts observed by Liebermann, 0. Fischer and others justify us in concluding that the chromophore is the most important factor in the destruction of fluorescence in colored substances. The fluorescing leuco-compounds differ from the absence non-fluorescing dye-stuffs only in one thing-the of the chromophore. The chromophore is consequently the cause of the different behavior of the two classes of compounds. The chromophore causes the partial displacement of the absorbed rays from the ultra-violet to the visible spectrum, possibly also the appearance of new absorption bands, and it causes a reflection of part of the visible spectrum. This reflected part is the one which weakens or destroys the fluorescence when complementary to it. Great stress is also to be laid on the first point, on the displacement of the absorption bands from the ultra-violet to the visible spectrum, especially in chromophores with high refraction and dispersion constants. The greater the displacement towards the red end of the spectrum the less is the possibility of fluorescence. By heaping up chromophores, the absorption bands can be displaced into the ultra-red, and fluorescence becomes impossible. The chromophoric groups therefore work against fluorescence in two ways. A different behavior is shown however by those compounds in which the absorption bands are far out in the ultra-violet and are only brought into the region of the visible spectrum by introduction of groups containing double bonds. A solvent (Kundt) can also have something the effect of a chromophoric group and can displace an absorption band towards the red end of the spectrum, this displacement being greater the greater the dispersion of the solvent. This established relation between fluorescence and the dispersion of
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the solvent throws some light on the action of chromophoric groups. The double bond, characteristic of the chromophores, causes a marked increase in the dispersion constant as compared with a single or a triple bond (Bruhl), Since adjacent chromophores re-enforce each other, the heaping up of double bonds, so characteristic of complex chromophores, causes an enormous increase in the dispersion constants and also a marked displacement of the absorption bands towards the red end of the spectrum. Increase of dispersion, the phenomenon of color, and the weakening of fluorescence appear in general all to be consequences of a special, presumably denser, form of matter such as we represent to ourselves by atomic groups with double bonds. The fluorescence is affected markedly and in an unfavorable manner by the salt-forming groups‘ of the dye-stuffs as well as by the chromophores. This is intelligible because most of the salt-forming groups displace the absorption bands towards the red end of the spectrum (Schiitze’s bathochrome goups). Only a few, such as the amino group, cause a displacement of the absorption bands towards the violet end (Schutze’s hypsochrome groups) and these increase the fluorescence when no other countervailing factors occur. The salt-forming groups are especially destructive of fluorescence when they have an auxochrome nature and re-enforce the dye-stuff. St. v. Kostanecki2 has shown that the color of a compound is more intense, the nearer the salt-forming group is to the chromophore. Salt-forming group and chromophore re-enforce each other I n the paper which I have already cited many times, R. Meyer called attention to the changes in the fluorescence of a compound caused by the introduction of a n O H or NH, group. H e showed that in the xanthone group substitution in positions 3 and 6 is favorable to fluorescence, especially when both are occupied by O H or NH,; while substitution in position I has t h e opposite effect. Since Meyer says nothing about the possible causes of this isomeristii phenomenon, I have considered the subject from a different point of view. Ber. chern. Ges., Berlin.
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Gertyud Wokev
more, the nearer they are, the effect being similar to but not so marked as that of two chromophores. Two adjacent hydroxyl groups re-enforce each other in a like manner as is shown by observations on xanthones, flavones and dioxyanthraquinones. Just as with the chromophores, this re-enforcement usually weakens the fluorescence in two ways. First, as has already been said, because of an increase in the color which makes the fluorescence less visible and especially when the color and the fluorescence are complementary. Second, because the reenforcement of the chromophore by the salt-forming group or the mutual re-enforcement of two adjacent hydroxyl groups causes an increased displacement of the absorption bands towards the red end of the spectrum, thus decreasing the possibility of fluorescence. I n agreement with our theoretical predictions we see in Table I that all the xanthones, having hydroxyl groups next a carbonyl group, or two free hydroxyl groups in the ortho position show either no fluorescence or a very slight one visible only a t great dilutions. The fluorescence becomes more intense the farther the hydroxyl is from the carbonyl group. I n 2 oxyxanthone there is a slight fluorescence in sulphuric acid solution. In 3-oxyxanthone the fluorescence is stronger and is visible both in alkaline and in sulphuric acid solution. The maximum is reached not when one para position to the carbonyl group is occupied but when both are. With the oxyxanthones and oxyphenonaphthoxanthones the variation of the fluorescence with the position of the hydroxyl group can be used for determining the constitution. With the flavones we observe the same thing as with the xanthones, though here the often greenish fluorescence is less affected by the natural color of the solution than is the case with In these cases, as in the previously considered case of the chromophores, there is the same limitation and possible reversal of the principle for the case of absorption bands f a r out in the ultra-violet.
'
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the bluish fluorescence of the xanthones. Among the monoxyflavones the 3-oxyflavone,‘ with the hydroxyl in the para position to the carbonyl group, has a beautiful bluish fluorescence in an almost colorless solution. The a-oxyflavone, on the other hand, has only a faint green fluorescence while the I-oxyflavone, which has not yet been made, will probably not fluoresce. This conclusion is justified by the similarity between the xanthones and the flavones and by the behavior of the polyoxyflavones which contain a hydroxyl adjacent to the carbonyl group and which all show very slight fluorescence in comparison with their isomers. Thus, among the dioxyflavones, chrysene3 ( I -3-dioxyflavone) has no fluorescence, in marked contrast to its isomers with the exception of 3-4-dioxyflavone. Specially noticeable for marked fluorescence are those derivatives which have a hydroxyl in the para position to the carbonyl group. These are the 3.2’-dio~yflavone,~ and the 3.3’-dioxyflavone5and the 3.4’-dioxy flavone, all of which show a blue fluorescence. If a hydroxyl group be introduced into these compounds in the ortho position to the carbonyl group, I .3.3‘we have the following substances : I .3.2‘-trio~yflavone;~ trioxyflavone and I .3.4’-trioxyflavoneg (apigenine). Apigenine and the 1.3.2’-trioxyflavone have only a faint greenish fluorescence while the I .3.3’-trioxyflavone does not fluoresce a t all. Two free hydroxyls in adjacent position have a weakening effect on fluorescence similar to that of the ortho position of hydroxyl and carbonyl groups. Like the 3.4-dioxyxanthone, St. v. Kostanecki and Emilewicz : Ber. chem. Ges., Berlin, 31, 699 (1598).
St. v. Kostanecki, Tambor and Levi : Ibid., 32, 326 (1899). St. v. Kostanecki, Tambor and Emilewicz : Ibid , 32, 2448 (1899). St. v. Kostanecki and v. Salis : Ibid., 32,1030:(1899). j St. v. Kostanecki and v. Harpe : Ibid., 33, 322 (1900). St. v. Kostanecki and Osius : Ibid., 32, 321 (1899). St. v. Kostanecki and Webel : Ibid., 34, 1454 (1901). * St. v. Kostanecki and Steuerman : Ibid., 34, 109 (1901). St. v. Kostanecki, Tanibor and Czajkowski : Ibid., 33, 1988 (1900).
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which is red in alkali solution, the 3.4-dioxyflavone’ does not fluoresce. The 2.3’-dioxyflavone2 fluoresces green and changes into the non-fluorescent 2.3’.4’-trioxyflavone3 when a hydroxyl is introduced in the position 4’. A similar behavior is to be noticed with the 3.3’-dioxyflavone4 which has a beautiful blue fluorescence and with the 3.4’-dioxyflavone4which has a violet fluorescence. Both change into the 3.3’.4’-trioxyflavone5which shows only a faint green fluorescence. Lastly, it is to be noticed that luteoline,6which does not fluoresce, has two adjacent hydroxyl groups in addition to the hydroxyl near the carbonyl group. There are therefore two factors in this case tending to decrease the fluorescencee This is the place to mention an experiment which shows the bad effect of two adjacent ortho groups upon fluorescence. On subjecting resorcinol to the zinc chloride fusion I obtained a substance which fluoresced as powerfully as fluoresceine itself. On condensing pyrogallol in the same way a substanc. was found which did not fluoresce a t all. That a t least a pair of free adjacent hydroxyls remained intact in this substance is shown by its character as a dye-stuff. With cotton mordanted with metallic oxides (Scheurer strips), with wool and with silk it produced a grayish green color, quite different from that which pyogallol itself causes. ’ Very often the regularities may be upset by a hydroxyl group reacting with a neighboring phenyl group forming a nearly closed new ring system which, in many cases, has the same marked tendency to fluoresce as a completely closed system. It is well known that the closing of the ring ____-
St. V. Kostanecki, Tambor and G. Woker : Ibid., 36, 4235 (1903). St. v . Kostanecki and Blumstein : Ibid., 33, 1478 (1900). a St. V. Kostanecki and Schmitt : Ibid., 33, 327 (1900). 1. c. 5 St. V. Kostanecki and Rozycki: Ibid., 34, 3721 (1901). 6 St. v. Kostanecki, Tambor and Rozgcki : Ibid., 34, 3721 (1901). 7 With other colorless aromatic compounds, pyrocatechuic acid, for instance, I was able to show the formation of colored lakes. 1 2
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exerts only a sliiht influence upon the properties of a compound save when it is accompanied by a perceptible displacement of the atoms in space and a consequent change in the tension of the molecule. A typical instance of this is to be found in the flavanolesl whose marked fluorescence is less affected by substitution than is the case with the xanthones and the flavones. The explanation is that they act like a ring system in which two benzenes are connected by means of an interwoven 7-pyrone furan ring (cf. p. 390). Bow do the other fluorescing substances act? A glance over the examples in Table I1 shows that the fluorescing compounds with salt-forming groups generally contain two such groups, usually arranged symmetrically, one of which is as far as possible from the chromophore. Thus in the fluorescing acridines, pyronines, thiopyronines, thiodiphenylamines, phenazines, phenoxazines, fluoresceines (in alkaline solution), etc., one amino or hydroxyl group is in the para position to the nitrogen or carbon atom having the double bond, while the other salt-forming group acts also as a chromophore, at any rate with quinoid dye-stuffs and their salts. This latter salt-forming group does not count a t all as producing fluorescence but only as a chromophore. In fact it works against the fluorescence even when it is the otherwise favorable amino group, .because the effect of a chromophore is greater than that of a salt-forming group. For this reason, in most fluorescing dyes, only one of the salt-forming groups is of importance to the fluorescence. It is always the one which has no double bond and which is in the para position to a carbon or nitrogen atom having a double bond. If this salt-forming group is absent, the compound ether has no fluorescence or much less than the substance from which it is derived. Instances of this are phosphine (chrysaniline), aposafranine, the indulines, New-Blue and others. If it is the other salt-forming group which is absent, as in rosinduline, the fluorescence remains. The strong fluorescence of the unsymmetrical rosinduline is an argument against St, v. Kostanecki, l,ampe, Tabor and co-workers.
symmetry relations being of importance to fluorescence. Fluorescence is not due in some mysterious way to the presence of two salt-forming groups i n the “ fluoresceine position” because a single salt-forming group may be entirely equivalent to the two, provided it is in the para position with reference to the chromophore and has no double bond. The enormous increase in fluorescence, which R. Meyer has shown for xanthone when the two symmetrical positions 3 and 6 are occupied, is in favor of the view that the 3-6-dioxyxanthone, in contradistinction t o the other oxyxanthones, reacts in the enol form as oxyformofluoresceine and not in the keto form as a xanthone. The intense fluorescence of tetramethyldiaminoxanthone is to be explained in the same way. The ready oxidizability of the pyronines to this compound seems to me an argument in favor of the enol formula. Of the regularities discussed in detail for the xanthones and flavones, one seems to hold for all fluorescing substances, namely that fluorescence i s most marked w h e n a salt-forming group i s in the farthest position f r o m the chromophore. Whether the other rules are of general application unfortunately cannot be told until the other classes of fluorescing substances have been studied as systematically as the xanthones and flavones. Even thefluoranes arenot wellenoughknown to beused with safety. It is true that R. Meyer has shown for this group‘ that a maximum fluorescence is obtained when the salt-forming groups are in the “ fluoresceine position ” (with a hydroxyl therefore in the para position to the double bond) while the fluorescence is destroyed when they are in the hydroquinone phthaleine position. Meyer has also brought together instances from the literature wherein homologous resorcinols show the fluoresceine reaction and are therefore to be looked upon as fluoresceines. As to this last point one is certainly justified in assuming the formation of genuine fluorane derivatives for cresorcinol and orcinol and this is probably true for K. Meyer : Zeit. phys. Chem.,
24,
468 (1897).
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dioxy-o-xylene. On the other hand, there is strong reason to doubt the fluoresceine nature of the condensation products of I .z.6-dioxytoluene, 1.3.2.4- and I .3.+6-dioxy-m-xylene, and mesorcinol. As we are dealing with substances which have never been isolated, only the two following reasons can be urged for their general fluoresceine nature: First, the substances in question are formed in presence of phthalic anhydride and second, they show the fluoresceine reaction. The first reason is not conclusive so long as it is not proved that phthalic anhydride actually enters into the molecule of the new compound and that it is not a question of the condensation products of the resorcinol component. The proof for the participation of phthalic anhydride in the reaction has not been furnished; on the contrary it has been proved for resorcinol that the condensation product is formed in presence of sulphuric acid without any phthalic anhydride being there. From this follows that the condensation product of resorcinol, and probably the analogous one of xylorcinol, has not a structure like fluoresceine. One should also not lay too much importance on the second test, the fluoresceine reaction, because substances which have nothing to do with fluoresceine show a very similar fluorescence. How careful one must be in such conclusions from analogy was brought home to me by experiments which I will describe here because the compounds I obtained possibly stand in close relation to the condensation products cited by R. Meyer. During the preparation of resacetophenone there is obtained as by-product a red tar which contains a fluorescing unidentified dye-stuff in addition to the non-fluorescing resaceteine, first described by Nencki and Sieber, and made synthetically a few years ago by Bulow. The resemblance between the fluorescence of this unknown substance and that of fluoresceine made me think that possibly the compound had a structure similar to that of fluoresceine. This might have been the case if the carbonyl of the resacetophenon'e had taken over the properties of the carbonyl of phthalic anhydride
and had reacted with two molecules of resorcinol. My guess that the fluoresceine dye-stuff had the following constitution
seemed to be confirmed when I obtained a fluoresceine-like fluorescing condensation product by fusing resorcinol and resacetophenone with ZnC1,. As a check, however, I fused resorcinol alone with zinc chloride and obtained a red tar with exactly the same fluorescence. It is thus proved that resacetophenone does not take part in the reaction. It is quite possible that the condensation product is identical with the fluorescing dye-stuff obtained during the preparation of resacetophenone and it is also possible that it is a lower homologue of one or more of the condensation products obtained by R. Meyer. Guesses as to the constitution of these substances are of course useless so long as these compounds have not been prepared pure and analyzed. As a system can take up from the surrounding medium chiefly those vibrations corresponding in period with those which it possesses itself, the observation of the absorption of rays of definite wave-length or definite oscillation frequencies may give a clue as to the rate of oscillation of the smallest particles. All true benzene derivatives have absorption bands in the ultra-violet. * Consequently by a suitable substitution of atoms and groups one might expect to change the frequency of the vibrating particles of any benzene derivatives so much that these vibrating particles should cause either pulsations in the ultra-violet close to the edge of the visible spectrum or should absorb those of the visible spectrum. One would obtain fluorescing substances, or by adding chromophoric The breaking of one double bond is enough to destroy the ultra-violet absorption.
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Carbazolic acid Thiodiphenylamine
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TABLE11-(
Continued)
~
~
Name
Methylacridine Acridic acid Acridic aldehyde Acridine yellow Acridine orange Phenylacridine Benzoflavine Acridine orange R Phosphine Pyronine Formo fluoresceine Leuco base of rosamine Eluorane Fluoresceine (in alkaline solution) Eosine (in alkaline solution) Rhodamines and anisolines Thiopyronine Phenacines Phenosafraninc? . Rosinduline [I] Rosinduline [21 Toluylene red Magdala red Phenofluorindine Diphenyl fluorindine Phenoxazine Resorufine Iris-blue Base of Nile-blue Base of new blue Naphth anthracene Dihydroanthracene Dichloran thracene Anthracene Anthranol Oxyanthranol H ydroquinizarine Methylphen ylanthracene Phenolph thalidine An thraquinoline Phenanthrene .
Salt group
Fluorescelice
very strong
