The Indian Happiness Wart in the Development of Photodynamic Action Douglas C. Neckers Bowling Green State University, Bowling Green, OH 43403
Rose Bengal is a xanthene dye that appeared in the commercial literature in Schultz's Tables' ( I ) dated 1881. I t was first synthesized by Rudolf Gnehm2 along with two other hlue-red dyes for wool, Erythrosin and Cyanosin B, during the period of 1880-1884 when Gnehm was employed a t Offenbach and Geschwanden, the private Basel predecessor of what became CIBA in the late 19th century. Gnehm's name first appears in the literature in Berichte (2) mentioned there by Baeyer3 (3). Baeyer (4) was clearly upset by von Hoffmann (5) who struck a claim to Eosin by publishing a structure proof. In a polemic entitled "Zur Geschichte des Eosins" (4) Bayer heatedly establishes that Emil Fischer first synthesized Eosin in Strasbourg "during the (FrancoPrussian) War" as part of his PhD dissertation hut that they did not publish its structure because Badische Anilin und Sodafahrik in Mannheim was commercializing the dye. We owe all of the initial work on the xanthene dyes to Baeyer (Strashourg). Baeyer was responsible for gallein, fluorescein, and their many halogenated derivatives. Graehe (BaselI4 gave the names to xanthene and xanthone.
Baeyer's first discovery in the xanthene series was gallein, from pyrogallol and phthalic anhydride (3).
Gallein is intensely red in dilute alkali but becomes blue in more concentrated basic solution. It is of commercial value because in strong acid it cyclizes to coerulein, a blue togreen Graebe's contribution to Rose Bengal was the first synthesis of tetrachlorophthalic anhydride, which, with resorcinol, com~risesthe startine materials for its svnthesis. Gnehm's wori is clearly a derivative of Baeyer's though Gnehm never nuhlished or natented his work. Such was industrial nractice at the time. The four most i m ~ o r t a n fluorescein t derivatives of these mrly days are fluoreswin, Fusin, Erythroiin, and Rose Henud". \Urnnine is another name for thr disodium solr oifluorescein.)
Schultz's Tables of Dyes, 1881, were a useful early compilation in the dye and color business (see Rose ( 1 ) ) .
Much of what the author knows about Rudolf Gnehm he owes to Gregw Graf and CIBA-Geigy (Basel).Rudolf Gnehm left ClBA in 1895 and became the first modern President of the ETH-Zurich in 1900. He was the President of the Swiss School Teacher's Federation from 1900 to the time of his death in 1924and was extremely influential in Swiss academic circles. -~ Many SWISS chemslry sr40entsnave receweo financia assistance in ,n verstly days from scholarships from the Gnehmstifung Tn~s founaalton was establ8sheo n Gnehm's honor for h s service Gnehm was from the village of Schaffhausen,a few miles up the Rhine from Basei, and on the occasion of the 50th anniversary of his death the Schaffhauser Nachrichten (2 June 1974) published a biography of their famous son. Graf found this story in the Schauffhauser Nauchrichten and made it available to the author. ' Baeyer nao a re atwei) heateo lhterature argument wlth von hoffb mann aoo.1 !he discovery of Eosm Von hoffmann( 5 ) fvst p ~ ~sned ts saucture after 11 nad oecome a commercial dye an0 oased on a sample provided him by Martius. This occasioned Baeyer's response "Zur Geschichte des Eosins" in which he points out in no uncertain terms that he and Emii Fischer first synthesized Eosin and that Hoffmann is stepping on his toes by getiing into the act. Graebe enters the oicture in a iono oaoer in Annalen about chioroohthniic There is no a%& between ,~ -~ -~~ connection ~- ~~.~ -~anhvdride'l61 Graeoe and Gnehm even though Graeoe was a1 the University n Basel and Gnehm was emp oyed at Ihe Chemca m d A w of Basel IC BAI: Graebe must, however, have been connected to ClBA Thesedyes remain important in many applications even today (7). ~~
The xan thene dves are rina-closed triphen. ylmethanes and as such can he compared wiih member; in that series. Thus the structure of fluorescein dianion and phenolphthalein differ only in the bridging oxygen of the xanthene ring under conditions of identical pH. Fluorescein, made from resorcin01, is a xanthene dye.-Phenolphthalein, from phenol, is a triphenylmethane dye.
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Volume 64 Number 8 August 1987
649
mordant6 dye used in calico printing.
phenolphthalein that fluorescein was a xanthene and considered it to have the structure of the lactone I. However, the lactone structure could not ~ .r o.o e r-l vaccount for the red color of fluorescein in basic solution and the zwitterionic forms IIA and IIB were eventuallv settled upon as the structures of the red-colored fluoresckin. One can buy both the quinoidal form of fluorescein and its intensely fluorescent disodium salt commercially these days, though the nomenclature for these compounds is highly confusing. A summary of the forms of fluorescein as a function of pH presented below:
I t was an obvious step from gallein and pyrogallol to resorcinol and fluorescein (8).
Phenolphthalein followed in a few years (9).
The structure proof of the basic ring skeleton of the nhthaleins. "from ~ h t h a l i canhdvride", and which Baever Lndertookafter hesynthesized phenolphthalein, represents one of the most impressive examples of deductive experimental reasoning i n the early history of organic chemistry (9). Based on elemental analyses done entirely with his own hand and the newly discovered reactions of Friedel and Crafts ( l o ) ,Baeyer converted phthaloyl chloride and benzene to phthalophenone and ultimately to triphenylmethane. Since he was also able to convert phthalophenone to phenolphthalein, the structure of phenolphthalein was thereby related to triphenylmethane. This work is summarized below:
The xanthenes represented an entirely new kind of dye when first synthesized. The most obvious first use was to dye cloth. Fluorescein dyes wool and silk directly from an acid bath, but the yellow color is not fast; it is called a highly fugitive dye. Eosin, however, is widely used as a direct6 dye to wool and silk. The names Eosin (Greek eos, meaning dawn), Erythrosin (Greek erythros, meaning red) and Rose Benga17 are commercial names. Eosin came from Badische Anilin and Sodafabrik. Erythrosin and Rose Bengal, as we have pointed out previously, originated from Gnehm at CIBA. Nanthrnesilnd ph~haleinsfound other uses almost immediatdy. Their culor rhanye.; with pH led tu their extensive
Direct dyes are colors which affixto cioth directly. Though this review is not of dye chemistry per se, dye terminology will occasionally creep in. Dye processes have been known for centuries and mordant is an old dye chemistry word. The attaching of a coiored organic compound to a fiber involves exceedingly complex chemistry. The first dye known to the ancients for example, was indigo. indigo is a totally insoluble organic dimer in its colored form.it can be used to dye cloth not in the colored form but only in its leoco(Greek,white)form which is soluble in dilute base. If a cloth to be dyed is immersed in a vat of ieuco indigo and the cioth then exposed toair, oxidation produces indigodirectly on the surfaceof the cloth. Leuco indigo is therefore called a vat dye because the dye is applied in the vat even though the coior actually forms afterthe dye is removed from the vat. The word mordantcomes from still another ancient dyeing technology. The Turks discovered the red coior of madder root, alizarin, centuries before Christ. Early applications of the coior known as Turkey red prepared conon for dyeing by first soaking the cloth in rancid olive oil containing lime. Then a solution of a metallic salt such as aluminum sulfate was applied to the cloth surface and the cioth was steamed. The cioth so treated was called mordanted(Latin,to bite) because the dye, alizarin, would now fix to the cloth. In appiication the dye complexes to the cioth through the aluminum affixed to the cloth forming a complex salt called a lake. Bengal Ros (in English, Rose Bengal) is named after the red dye used by Bengali women to symbolize marriage (Indian Happiness Wan). The latter is either a root extract or cinnabar (HgS)depending on the source.
'
Baeyer reasoned by analogy after proving the structure of 650
Journal of Chemical Education
use as acid-base indicatorss. T h e i r photochemical propertit.5 were tliscovered in the n m t e x t ot' photudgnnmic artion, the lethal eilecr of sunlight and d y r s tm bacteria ( 1 7 ) . Near neighlwrs hare been and are s t i l l used i n spirit-soluhle d i t t o machine annlications. M u c h later soectroscooic orooerties such as the*i; fluorescence resulted i n t h e i r extensive applicat i o n as laser dyes.9 Phenolphthalein has strong laxative act i o n a n d is widely used in proprietary medicines. We shall concentrate in this review o n Rose Beneal. tetrachlorotetrai~douranine.~O Rose Bengal interested i s ;riginallv because it was so freauentlv emoloved as a source of ~ h i o u l d f i i d i t s structure in singiet oxygen (18). ~ h o u one o l d organic textbooks such as Noller (20) a n d Fieser ( 2 0 , i t
. . .
.
8The use of dves as indicators for acids and bases traces throuoh the early history of tne tr~phenylmethanesand the xantnenes. The reader is best referred to hammen's class c work ( 1 ll for tne empwical oelail that was applied lo finding md cators which would change color over narrow pH ranges. The indicator action of the xanthenes and the phthaleins is indigenous to high school chemistry courses and inorqanic aualitative and quantitative analysis. Phenolphthalein is red in dilute base and colorless in acid and concentrated sodium hydroxide. At the time students are introduced to indicators, however, they are unable toconsider theorganic chemistry of the dyes because they do not have the background, and this part generally gets left by the wayside forever; phenolphthalein is an obscure organic dye. Indicators were used to distinguish acids from bases from the discovery of the phthaleins and the xanthenes. Wiihelm Ostwald (Die wissenschaftlichen Grundlagen der analytische Chemie) suggested indicators to be weak acids or bases whose colors differed in the ionized and the un-ionized forms. Thus an indicator dissociates ( 12. ~~~
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was readily apparent t h a t n o structural o r synthetic work h a d been done o n the dye in nearly a century. W e thought this strange when considered in the context o f the many physical organic a n d spectroscopic studies in which Rose Beneal was used as a t r i n l e t sensitizer. Our i n i t i a l efforts werevtherefore simply t o rkdiscover some of the chemistry of Rose Benaal hut t o d o so usina contemoorarv instrumentation.'l'hr latter is more i n promise than in practice, hower,er, rince R w e Hen:nl has a molecular weight of 1017, is soluble in only very polar sol\wn-preferably water- -and has only twu hydrugens. A d d to that an extinction cocificient ot'over 100,000, four or f i w different structures depending i m the pH of thesi~!ution,and t h r c e r e a d i l v m a n i p u l s r ~ dfunrrional group;, and one h i s an oryanir molecule that requires very +killed experimentati~ntbeforr i t reveals m u c h abuut itself. I h e Renaal is s\.nthesizril from chlorouhthalir anhvdride a n d resorc&ol. E h l o r o p h t h a l i c a n h y d i i d e derives" f r o m Graebe (6) too,
-
~
~
W,
and [Htl[I-1 K", = [HI]
-
This exoression Dredicts how indicator color chanaes with OH.Structural theor es of nnacator act on ( 12, 13 proposeds~multaneously and ndependently tnal phenolphtnale n emsled n acndas the lactone form and n base as the quano dal form Change of color. In tnls theory, was accompanied by a change in chemical constitution. The theory of indicator action is reviewed clearly in Rosenblum's translation of I. M. Koitoff's book iI ¶ . (Koltoff. of course, was Professor of Analytical Chemistry at the University of Minnesota for many years.) Morrison and Boyd ( 1 5 ) captured the theory of weak organic acids and bases in their 1959 textbook. Unfortunately they totally missedthe organic chemistry of Baeyer's dyes and indicator actionthe understanding of which gave rise to the theories which begat organic chemistry, and the understandingof organic acids and bases. in the first place. The commercial importanceof indicator action is immense-even today. Triphenylmethane acid-base chemistry is solely responsible for the color chemistry associated with the carbonless carbon paper industrv and some of the earlv exoeriments of Emii Fischer and his cous n On0 F scher, on maiach te green and crystal vloer lactones have Dew respons~blefor chongmg the en1 re course of ndustroal growth in this field.
and a n 1885 C I B A patent teaches i t s synthesis from phthalic
ether solution in a urea-formaldehyde microcapsule and laid on the surface of a paper sheet. A second so-called base sheet is coated with an acid developer. Pressure on the microcapsule with a sharp object such as pencil breaks the capsule and releases the dye precursor on the acid surface where the lactone undergoes ring opening and the purple color of crystal violet appears. The lactone form of the dye and the ring-opened form are in a tenuous equilibrium controlled by the polarity of the medium. in the early days of carbonless carbon paper technology, the base sheet was coated with an acid clay that was essentially a polar, ionic medium for the dye. Addition of water to this medium drove the highly organic dye back into the nonpolar solvent where it reverted to the more nonpolar lactone form. Thus. with acid clay developers carbonless carbon paper copies were not "archival'', that is, stable over long periods in humid environments. This problem was circumvented in the early '70's by replacing the acidic base sheet surface with a iow-moiecular-weightNovalak resin. Novalaks are phenol-formaldehydes and as such have both a polar component, the phenolic OH, and a nonpolar component, the aromatic backbone. This combination of factors apparently provides a sufficiently nonpolar environment for the ring-opened dye, and water has much less of an effect on it. Orlo Flscner ( 16) polnled OLI lhls phenomenon. The amhor first encountere0 Fiscner's paDer when it was introduced as evidence in a major carbonless carbon paper patent nfringement litigation n 1984. "art of the reason dye chemistry is so mystifying to the contemporary student of organic chemistry is because experiments to make new colors preceded by many years the structural theories and systems of nomenclature that we can now use to understand the experimental observations. The Rhodamines, so named, are "red amine" xanthene dyes made from aromatic amines and phthalic anhydride. Rhodamines B and 6G were by far the most popular xanthene dyes for coloring cloth because their colors were so sharp. This is the result of their fluorescence, which, in turn, is responsible for their application in dye lasers. 'O The Chemical Abstracts far Rase Benaal - - - name ~. - - - -- is- 4.5.87-tetra.~.~. chloro-3'.6'dlnydroxy-2'.4'S1,7 '-tetraiodosp r0[isooen~ofuanyl~3rlj. 9'-[9H]xanlhenl-3-one 0 sodium salt. Tnis name is wrong. It ,s the name of the lactone and is based on the nomenclature for fluorescein, which is, unfortunately, based on 19th century organic chemistry. Rose Bengal does not exist as the lactone in commercial samples. it exists as the disodium salt of the xanthenedye. Naming thedye on the basis of the lactone is erroneous and irrelevant. In fact that lactone of Rose Bengal was not reported until the work of Lamberts and Neckers ~
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Crystal violet lactone is the colorless precursor employed in carbonless carbon paper technology today. It is encapsulated in diphenyl
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(19). Volume 64
Number 8
August 1967
85 1
Figure 1. (let0 Spectrum of Rose Bengal (dilute solution).(right) Rose Bengal (conc. solution).
anhydride and antimony pentachloride." I.
UOi.
NaOH
a ON.
Rose Bengal
Rose Bengal is red in base and is about the same color in dilute solution as the decorative Indian h a.~.~ i n ewart s s color worn by Bengali women at the center of the hairline to symbolize marriage. This was the source of its name. There are two major absorptions due to the xanthene chromophore at 530 nm and 558 nm, approximately. The wavelength a t which these two absorptions are found is a function of solvent. Rose Benaal absorbs a t the longest wavelengths in polar, nonhydroxylic solvents. ~ y d r o & nbonding reduces the A,. ... by. afew nanometers. The relative peak heights are a function of the extent of ionization at C-6 as well as the concentration of dye in solution. Most highly absorbing dyes do not follow Beer's Law a t concentrations above about M as dye molecules tend to aggregate in what is loosely called in the literature a dimeric dye. The dimer shows up in that the shorter wavelength of the two absorptions increases in height relative to the longer one. The spectrum observed for a dilute solution of Rose Bengal is shown in Figure l a , that for a more concentrated solution in Figure lh. Two changes may occur in the absorption spectrum when the pH of a Rose Bengal solution is decreased. Protonation at C-6 produces the spectrum of the quinomethine system in the absence of delocalization (Fig. 2); and lactonization a t C9 wipes out the color entirely. These changes also affect ~~~
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solubility. The lactone, in water or methanol solvents in which they dye, itself is soluble. Lactonization provides a simple procedure to purify the dye. Most impurities in the commercial sample are inorganic salts, and precipitation of the lactone with concentrated HCl leaves these impurities in solution. Further purification can be effected by recrystallization, either of the lactone, or preferably of the lactone diacetate. There are many other methods r e ~ o r t e dthat claim to affect purification of Rose Rengal.l'h&t. mc,~hodsarealmost mrir& chromatographic and t.scnti.~llyuseless.l! Thin-layer chromarograms of Ruse produce three red spots of e&entially the Bengal same color, none of which properly analyzes for tetrachlorotetraiodouranine. In addition to the inorganic salts, other impurities include halogenated xanthene analogs with either fewer or greater numbers of iodine atoms. Elemental analysis for carbon provides the simplest test of purity. Rose Beneal is ~ r o d u c e dtodav" hv" both J a ~ a n e s eand Indian cwnpanies. The rvmmerc~alsynthesis is idvntical to the Baever s~nthrsi;. Reicminol and rrtrachl~mohrhalic anhydride areheated with anhydrous zinc chloride to about 180 O C . The cooled melt is treated with NaOH or KOH and the product iodinated with an alcohol solution of 12. The extinction coefficient of the dye is used as the commercial measure of purity. An American company, for example, which once made Rose Bengal, still offers the remainder of
" Schenck, in an early paper (22), mentions puritying Rose Bengal by means of the lactonediacetate;however, the procedure was never
published. Schenck's later definitivepapers on dye-sensitized photooxidation (for example, G. 0.Schenck and K. Gollnick, "Ueber die schneilen Teilprozesse photosensibilisierter Substrat-Uebertragu* gen': Forschungsbertichte des Landes Nordrhein-Westfalen,#I2561 simply resort to extracting the dyes with organic solvents. This is an essentially useless procedure because the impurities are all inorganic salts. Chromatography is a mess; but some papers suggest ways to use it (23). 652
Journal of Chemical Education
Figure 2. Rase Bengal spectrum acid solution-no lactonization.
an old hatch of dye which analyzes as 85% in dye content.12 For purposes of simplification, we have elected to represent only the reactive functional groups of Rose Bengal in a shorthand notation. The fully developed dye chromophore is in the form of the bis anion, and one can esterify both C-2 and C-6 under appropriate conditions. Unlike less sterically hindered xanthene dyes, Rose Bengal does not react with alkyl halides at C-6 and ethers of the dye are unknown.
Rose Bengal is esterified at C-2' by reaction with alkyl halides in either DMF or in acetonelwater (50:50). The henzyl ester formed under the latter conditions is red in polar solvents. orange " in nonwolar solvents. This is a characteristic color change resulting from ionization of the protonated form that forms from the acid produced in acetone water at C-6. The red color is essentially identical to that of the dianion indicating that ionization at C-2' has virtuallv no effect on the observed absorption spectrum. The C-2'h&yl ester cannot he esterified with acetic anhydride (19) since the henzyl group leaves as the benzyl carhocation under these conditions and one forms the lactone diacetate. This ionization of the henzyl group a t C-2' is assisted hy the electropositive C-9. The ethyl ester can he esterified a t C-6 with acetic anhydride. The C-2' ethyl ester, C-6 acetate, is orange in all solvents including polar solvents like methanol and ethanol.
Emission Spectra
Fluorescein is reputed to be the most fluorescent comoound. oer unit weieht. known. Its beautiful ereen fluorescence in alkali is stir1 dktectahle to the nakedueye a t 1 part per 40,000,000. This fluorescence is self-quenched and a t higher concentrations fluorescein solutions appear orangish yellow. Fluorescence depolarization using fluorescein is one of the most useful contemporary medical diagnostic tools because of this high sensitivity. Using this technique one measures the emission of fluore&ence &om a hiologiial molecule to which fluorescein has been immobilized. Under conditions where the biological molecule is not modified by its surroundings, the fluorescence has a certain polarization. If a hioloeical interaction such as the reaction of an antibodv with its antigen occurs, the polarization of the fluorescence is decreased. This can be auantitated and deoolarization of fluorescence can he used to count the specific antigen concentration in the serum under investigation. The four iodines of Rose Bengal inkease its intersystemcrossing vield relative to fluorescein. This means that Rose ~ e n ~ a l f l u o r e s cred, e s hut weakly. The extent of ionization a t C-6 influences the observed emission spectrum. The spectra of the his-ammonium salts show this clearly (Fig. 3). The shorter wavelength emission is that of the form non-ionized at C-6. The relative spectroscopic characteristics of fluores-
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The X-ray crystal structure of Rose Bengal lactone shows several interesting features. The lactone ring is perpendicular to the xanthene; the C-9-0 lactone hond is about 20% longer than is the ester oxygen hond of a typical five-membered ring lactone; and the central dihydropyran ring of the xanthene skeleton is puckered out of plane enough by the iodines to make Rose Bengal lactone nearly colorless. The lactones of Erythrosin (orange) must have a much more planar central dihydropyran ring. l2 Analysis of dye content is done on the basis of the extinction coefficient at the maximum absorption and has nothing to do with the chemical composition of the dye. A dye sold at 85% dye content simply has 85% absorbance per unit weight of some standard absorbance per unit weight reported in some standard literature table. In many cases the standard is old; in many other cases-and this is the case with Rose Bengal-thedye chemistry being used to produce the dyes in commerce is from the 19th century. What this obviously means to the contemporary chemist is that dyes as obtained from suppliers must be treated with a great deal of experimental skepticism. The name on the bottle and the color in the bottle may be standardized, but to relate a structureto that name is tenuous at best.
Figure 3. Electronic absorption spectra of Rose Bengal derivatives.Top, Rose Bengal dipiperidinium salt in methylene chloride. Bottom, Rose Bengal, disodium salt in MeOH.
Volume 64
Number 8
August 1987
653
The Photophysical Properties of Fluorescein and Its Halo Derivatives Compound Fluorescein
Eosin Erythrosin Rose Bengal
An ,m 491 514 525 548
4F
4ST
410,
$ST
0.93 0.63 0.08 0.08
0.03 0.3 0.6 0.76
0.1 0.4 0.6 0.76
0.32 0.69
0.03 0.88
cein, Eosin, Erythrosin and Rose Bengal are given in the table. Photochemical Properties and Photodynamic Action Pbotodynamic action, the lethal effect of light and oxygen on microbes in the nresence of certain dves, was discovered at the turn of the century by Raab and v o n ~ a p p e i n e (1 r 7). In the very long paper by von Tappeiner and Jodlbauer (17) on the effect of dyes, light, and oxygen on paramecia puhlished in 1904, all of the known derivatives of fluorescein were studied as photosensitizing ilyes including Kryrhrosin and Rose Rcnml. One of the interesting observations \on Tappeiner makes is that there appears to be an inverse relationship between the fluorescence of the dye and the photodynamic effect. This 1904 paper is the first evidence in the literature that the xanthene dyes have interesting photobiochemi~trv.'~ Singler oxygen rhemistry has been reviewed both criticalIv (251and historicallv (26).The iirst roof that dvcs served aJsensitizers in synrheticphotoxygenarions was reported hv \Vindausand Hrunken (271who is&ited eruosterin peroxide from the ~osin-sensitizedoxygenation of eigosterin. Kautsky's experiments (28) supported, and he first theorized the existence of, a metastahle state of oxygen in dye-sensitized photooxygenations. In the salient experiments Wirstellengetrennt zweiKieselsaureadsorbate her,das eine uon einem fluareszierenden Farbstoff k. . B . Trvoaflauin).das andere rwn eihern A k r ~ p r o rt z . 0 p - ~ r u k o n r l i l l n j . ' ~ b ~ u idred e n durrh perne,nornt,l I'errciben i n n i ~~~crrn,vcht. A u l d m e l l ' w e rind S~naibilisolurund Arreplor rournltrh ordlknrnn~mw n u i n o n d u r getrennt.
..
o-Leucoaniline. a trinhenvlmethane dve with no ahsomtion in the visible; absorbedon solid parkles, was oxidized to Rosaniline (called also fuchsin) when absorbed Trypaflavin dye was irradiated in the presence of oxygen. This clever exoeriment. similar to the Paneth exoeriment that demonstpated the'presence of free radicals i n the gas phase, presented the first evidence that a metastable state of oxygen was carried through solvent from the sensitizer to the acceptor even in the absence of molecular contact. Kautsky's theories that there was a metastable state of oxygen transmitted through solvent from donor to acceptor nn&hat singlet oxygen wis that metastable state mer with n lor of skepticism. Gaffron (29) and larer Srhenck (30) were largely responsible for this opposition. They believed that the sensitizer participated in the oxidation step and had to he involved in the mechanism of the reaction in the form of a dye-acceptor complex. Their arguments centered on the apparent influence of the structure of the sensitizer on the nature of the reaction products. The Schenck-Gaffron view was largely accepted by the photochemical community, and Kautsky's theories were discredited to the point that Kautsky was never able to obtain a proper academic position. In fact, the Schenck view prevailed until the '60's when Foote (31) and Corey (32) provided conclusive evidence for the existence of singlet oxygen in dye-sensitized oxidation pro654
Journal of Chemical Education
cesses hv directlv comnarine of these nrocesses . -the oroducts . with thbse forked from processes known to fork singlet oxygen. In Foote's case a chemical synthesis of singlet oxygen was used. Corey and Taylor used amicrowave discharge. Schenck's group (33) was responsible for the acceptance and general popularizing of Rose Bengal as a dye sensitizer for singlet oxygen formation. According to former Schenck students "it was always around the lab" (34). Schenck's experimental work was based on that of Kautsky, of Gaffron (291, and of von Tappeiner. The synthesis of ascaridole, which Schenck carried out for practical reasons usina spinach leaves as the source of sensitizer during the latterstages of World War I1 (35), was reported with his mentor Karl Ziegler in 1943. The 0-0 band of the Rose Bengal triplet state lies 39.5 kcallmol ahove the ground state. The 0-0 band of the Rose Bengal singlet state lies 45.0 kcal/mol ahove the ground state, and the fluorescence hand is structured as the mirror image of the absorption band. The quantum yield of intersvstem crossing. -. &,. . is virtuallv"eaual . to the auantum vield fbr singlet oxygen formation; the quantum for fluorescence is about 0.05; the remainder of the enerev is wasted in nonradiative processes (36). The Rose Bengal triplet is entirely quenched by dioxygen in solution and in a diffusion-controlled process. Singlet oxygen (A'g = 23.5 kcallmol above ground) is formed almost entirely from the quenching, hut there is a small amount of superoxide radical anion (37) as well. Singlet oxygen formation from a dye is studied quantitatively by a relative actinometric method developed originally by Schenck and Gollnick (36). In this method, a chemical trap is employed that traps 100% of the singlet oxygen formed in a diffusion-controlled Drocess. If the sensitizer is present in sufficient concentration so that all the incident light is absorbed, the efficiency of the process and the rate of singlet oxygen formation is represented by the first-order disappearance of the trap. Schenck and Gollnick (36) . . found the quantum yield of singlet oxygen formation for Rose Bengal to be 0.76 in methanol, and one uses this value as a standard for mensuring the quanrum yieldsof s~nyletoxygen furmarion from other similar sensitizers. The kinetic parameters for singlet oxygen formation by Rose Bengal derivatives depend on the quenching parameters of the acceptor and on the solvent (37,38). Rose Bengal bleaches when irradiated in the presence of oxveen. Thoueh the hleachine orocess has never been studied &antitati;ely, it is fast en&h so that Rose Bengal must he periodically replenished when it is used as a sensitizer for synthetic oxidations. Bleaching is typical for most dyes, however, and Rose Beneal - is neither the best nor the worst in this respect. The bleaching process involves one of several possible reactions that remove the chromophore. The most likely of these is peroxide formation across the quinoid, or A ring. Evidence for this is presented by the observation (39) that the bleaching reaction can he reversed with reducing agents such as borohydride. In the solid state, evidence has recently been obtained that points to radical cation and radical anion formation as the initial step in the bleaching ethyl ester in a thin film; the
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l 3 Von Tappeiner and Jodlbauer (17). The earliest extension of von Tappeiner's result to human physiology is that of F. Meyer Betz (24) and this work is responsible for what has become today phototherapy. Meyer Betz reports selfinjecting 0.20 g of hematoporphyrin (a singlet oxygen sensitizer) in 300 mL of a saline solution. He then spends the next several days in and out of the sun determining what effect the dye is having on him. The paper makes interesting reading for those with strong stomachs.
major products from the photochemistry are radical cations and radical anions below.
Radical cation and radical nnion formation is a likely preredent to rwction with oxycen and formation of the bleached Rose Bengal derivative Polymeric dhivatives of Rose Bengal were developed in 1973. The orieinal idea was based on Merrifield's work (41. 421, and polymer Hose Bengal u,nssynthesized asa heterogeneous source 01 s~nrletoxwen ( 4 3 ) bv Xeckera and Hlossev. In a collahoration~with"&e ~ e c k & sgroup, Schaap add Thaver found ~ o l v m e Rose r Benaal to be a useful sensitizer for singlet oxygen-formation. ~ol;mer Rose Bengal, marketed later as Sensitox (44).was the first example of a heterogeneous energy-transfer donor that did n o t degrade phGochemically during the process of its use. The quantum yields of energy transfer from Rose Bengal immobilized on Merrifield heads have beeli studied extensivbly by Paczkowski (45). Based on information obtained from ethdies of soluble Rose Bengal polymers, dye molecules immobilized on beads are located a t sufficient distance from one another to he essentially site isolated. There is no evidence in the spectra of the beads or from energy-transfer experiments that the immobilized dyes interact with one another. Thus. the Quantum vields of sinelet oxvaen formation from polymer ~ o s eeng gal heads areahoutthe same as observed for a soluble derivative of Rose Benaal in the same s
