Edgar Buckingham: Fluorescence of Quinine Salts

Bull. Hist. Chem., VOLUME 27, Number 1 (2002) 57

EDGAR BUCKINGHAM: FLUORESCENCE OF QUININE SALTS

John T. Stock, University of Connecticut

Malaria, an often-fatal disease, has been a worldwide factured from cinchona trees that are cultivated in South plague for several thousand years. The discovery of America and in the Far East. the efficacy of substances present in the bark of vari- It must have been known ous cinchona trees, native since ancient times that certain to the Andes, provided substances appear to have one some relief. A real anti- color when viewed by transmit- malarial drug was not ted light and another when available until 1820, when viewed obliquely. Mineralo- Joseph Baptiste Caventou gists recognize a type of fluor- (1795-1877) and Josephe spar, pale green when viewed Pelletier (1788-1842) iso- against the light, but appearing lated quinine from the blue when viewed at an angle bark (1). Eighty years af- to the light. Unrefined petro- ter their discovery, a statue leum shows the same kind of honoring these chemists effect, as do certain substances was erected in Paris (Fig. when in solution. Fluorescein, 1). used both in the laboratory as Other workers estab- an indicator and industrially for lished the formula for qui- the location of leaks in waste nine, showed that it acts as water systems, is a familiar ex- a diacid base, and that it ample. Another is quinine or, is a methoxy derivative of because of its low solubility in a companion alkaloid, cin- water, one of its salts. The so- chonine. The elucidation lution, colorless when viewed of the structure of these directly, appears blue when compounds, largely due to viewed at an angle to the inci- the work of Wilhelm dent light. The phenomenon Figure 1. Caventou and Pelletier Königs (1851-1906) and exhibited by these various sys- Paul Rabe (1869-1952), was finally published in 1908 tems is termed fluorescence. With modern laser instru- (2). More than three decades passed before the partial mentation and highly sensitive detectors, fluorescence synthesis of quinine was achieved (3). The first com- has become a powerful analytical technique. For ex- pletely stereoselective, total synthesis of this compound ample, the laser-induced fluorescence detection of was reported in 2001 (4). However, despite the discov- derivatized angiotensin peptides is applicable to quan- ery of other antimalarial drugs, quinine is still manu- tities as small as a few hundred zetamoles (5). 58 Bull. Hist. Chem., VOLUME 27, Number 1 (2002)

The systematic ted at a wavelength longer than that of the incident beam, study of fluorescence became known later as Stokes’ Law. was initiated by As- Stokes noted that the quinine salts of numerous tronomer Royal, acids exhibited fluorescence, exceptions being the salts John Frederick of HCl, HBr, and HI. In fact, the addition of one of Herschel (1792- these acids to a fluorescing quinine salt solution de- 1871) (Fig. 2). He stroyed the effect. However, the fluorescence returned named the phenom- when the interferent, or quenching agent, was removed; enon epipolic disper- e.g., by treatment with HgO. Mercury halides did not sion, derived from quench the fluorescence. the Greek for “surface,” because he be- Other workers, notably Victor Pierre (1819-1886) lieved that the effect (11), Jacob Edward Hagenbach (1833-1910) (12), and originated in a layer Cornelius Joseph Lommel (1837-1899) (13), extended adjacent to the en- the study of fluorescence. Pierre showed that a given trance of light. For Figure 2. John F. W. Herschel substance does not fluoresce if the wavelength of the Herschel and his suc- incident light is greater than a certain minimum. cessors, the usual light source was the sun or daylight. Hagenbach examined the fluorescence of numerous, Because the “detector” was the eye, the observations mainly organic substances, including quinine sulfate. He were not numerical but merely comparative. Colored found that the spectrum of a solution of this salt exhib- glass or sometimes a prism was used to select spectral ited two maxima. One of Lommel’s discoveries was portions of the incident or emergent light. Observations that the fluorescence radiated by a volume element of a had to be made in a dark room or enclosure, with the substance is proportional to the amount of the exciting incident light entering through a hole or slit. light absorbed. In 1845 Herschel described his experiments with This was approximately the state of affairs when solutions of quinine tartrate (6,7). He noted that when Edgar Buckingham (Fig. 4) began his work on fluores- an approximately 1% solution is placed in a tall glass cence, particularly that of quinine salts. Born in Phila- before an open window, the blue color can be seen by delphia on July 8, 1867 and graduated from Harvard in looking down into the glass. When the solution was 1887, Buckingham spent a period in Stra_burg before trickled from one glass to another, the thin film seemed moving to Leipzig in 1890, where he began the work to be equally effective as the bulk solution. Herschel mentioned. His aims were to extend the then-known commented that light transmitted through a “quiniferous facts and to interpret the results in terms of the Arrhenius solution,” thus producing a “dispersion,” did not pro- ionic theory, which was duce a dispersion in a second portion of solution. strongly promoted in In fact, the phenomenon described by Herschel had Ostwald’s laboratory. been noted earlier by David Brewster (1781-1868) (Fig. Buckingham’s optical 3). He used a lens to focus sunlight and was able to equipment was simply demonstrate that the “dispersion was not confined to the the Stokes dark box, surface layer, but extended well into the solution (8).” with colored glass fil- ters for sunlight and, George Gabriel Stokes (1819-1908) greatly ex- occasionally, artificial tended the observations of Herschel and Brewster. With light (14). The avail- a box-like enclosure that enabled him to observe with- ability of electrolytic out darkening the room, Stokes examined quinine sul- conductance apparatus fate, solutions of various plant extracts, certain glasses, was a major asset, al- and even uranium compounds (9). He thus demonstrated lowing him to assess that many systems exhibited phenomena similar to that the ionic state of his so- shown by quinine salts. In a later report. Stokes used lutions. “fluorescence” to replace the older term (10). An apparently universal effect, that the fluorescence was emit- Figure 3. David Brewster Bull. Hist. Chem., VOLUME 27, Number 1 (2002) 59

Preliminary experiments were carried out with eosin + + 2- Ch.H2SO4 ChH ++H SO4 (tetrabromofluorescein), which behaves as a dibasic acid. Its red solution exhibits a green fluorescence. These The addition of alkali destroyed this cation and also experiments convinced Buckingham that the intensity the fluorescence. However, the fluorescence was in- of the effect was governed by the concentration of the creased by the addition of HNO3. Another possibility ions of the solute. He then turned to quinine, known to considered was that the salt dissociated as follows: contain two basic nitrogen atoms and thus capable of Ch.H SO 2+ 2- forming two series of salts. Because the basic proper- 2 4 ChH2 + SO4 ties of quinine are weak, however, Buckingham pointed out that, with respect to electrolytic dissociation, the salts From an extensive series of measurements of elec- formed with one equivalent of acid per molecule tend trolytic conductivity, Buckingham argued that, in solu- to act like binary neutral salts such as KCl. tion, quinine hydrogen sulfate partially dissociates to + 2+ give both univalent (ChH ) and divalent (ChH2 ) cat- Buckingham experimented with the hydrochloride, ions: acetate, monochloroacetate, nitrate, and sulfate of qui- + + 2- 2+ 2- nine, as well as with the hydrogen sulfate, which he Ch.H2SO4 ChH ++H SO4 + ChH2 + SO4 termed “bisulfate.” With approximately millimolar solutions of each of the salts, he found that the hydrogen Buckingham came to the conclusion that the fluo- sulfate fluoresced more strongly, the acetate less strongly rescence was due to the quinine cations, and that the than the other salts, excluding the hydrochloride. As divalent species was the more effective. The addition had been found by Stokes, the hydrochloride was of HNO3 (i.e., of hydrogen ion) to a solution of nonfluorescent. From the German, “Chinin,” Ch.H2SO4 favors the conversion of the univalent to the Buckingham used the contraction “Ch” to indicate this divalent cation, with corresponding increase in fluores- compound. Quinine hydrogen sulfate was thus written cence. Conductometric measurements of the quinine salts of strong acids (other than HCl, etc.) at millimolar Ch.H2SO4, or (Ch.H).HSO4. The fluorescence of a solution of this salt decreased as KOH was added, vanish- concentration indicated almost complete dissociation. ing when the molar concentrations of this salt and of The stronger fluorescence of the hydrogen sulfate thus added KOH had become approximately equal. Because cannot be due to dissociation greater than that of the of the low solubility of free quinine in water, the experi- other salts. ments were conducted in approximately 64% alcohol. If the above explanations are correct, the addition In molecular terms, the reaction was presumed to of excess strong acid to equimolar solutions of the vari- be: ous univalent quinine salts should cause the fluorescence to rise to the same maximum. Buckingham proved this Ch.H2SO4 + KOH Ch+ H2O + KHSO4 experimentally (halides excluded) and found that even weaker acids in greater excess were also effective. He This implied that free quinine does not fluoresce. In a also found that if small amounts of HNO3 were added possible alternative reaction: to a millimolar quinine sulfate solution (cation, Ch.H+), the conductance decreased. Because HNO is an excel- K SO 3 2Ch.H2SO4 + 2KOH Ch2.H2SO4 + 2H2O + 2 4 lent conductor, this seems surprising. This result was attributed to the conversion of the univalent to the diva- the (normal) sulfate, (Ch.H) SO , would be formed; but 2 4 lent quinine cation, with consequent removal of the the absence of fluorescence implied that the normal sul- highly conducting hydrogen ion: fate was also inactive. However, a 5mM 60% alcoholic solution containing both KOH and the normal sulfate Ch2H2SO4 + 2HNO3 ChH2SO4 + Ch.(NO3)2 produced a distinct, if weak, fluorescence, thus suggest- ing that the reaction indicated by equation (2) was un- If only the cations are considered, the equation be- likely. comes:

Because quinine acts as a univalent base in aque- + + 2Ch.H 2+ ous alcoholic solution, Buckingham assumed that the 2Ch.H + 2H 2 + fluorescent species was the cation ChH , formed by the Obviously, the amount of hydrogen ion (i.e., of reaction: HNO3) added must be less than that implied by equa- 60 Bull. Hist. Chem., VOLUME 27, Number 1 (2002) tions (6) and (7). the Table below, is similar to that of the lyotropic series Buckingham demonstrated of ions that is relevant to various physicochemical phe- this experimentally with nomena such as the precipitation of colloids. amounts of HNO3 that ranged from 1/5th to 1/ Refractive index measurements were used to ob- 50th of the amount of qui- tain the numbers beneath the symbols. The numbers nine nitrate. Through the are measures of ionic deformability, i.e., the looseness common-ion effect, the of the binding of the outer electrons. The authors sug- addition of K SO to a so- gested that the high deformability of the halide ions 2 4 - lution of quinine sulfate (CNS , known to be a powerful quencher, was not evalu- might be expected to de- ated) enabled the excited quinine cations to return to press the dissociation of normal conditions by radiationless transfer of energy the quinine salt, and thus through collision with the halide ions. diminish the fluorescence. Figure 4. Edgar Buckingham The work of Francis Perrin (1901-1992) was quoted In fact, the latter increased, in support of this collision theory. He showed that as a fact attributed to partial conversion of Ch.H+ into di- the viscosity of the solvent is increased, a greater con- valent Ch.KH+ ions. centration of the fluorescing solute is needed to obtain Finally, Buckingham turned to the well-known maximum emission (17). Presumably the frequency of quenching of fluorescence by halide ions. He repeated collision between the quinine ions, and hence their acti- the experiments by Stokes, looking for possible causes vation, is diminished in a more viscous medium. This of the effect. Halide solutions absorb active portions of decrease should also apply to collisions between the qui- the incident light or the fluorescent light itself. How- nine ions and the quenching ions. The diminution of ever, this light from a quinine salt solution was not ex- the quenching power of the halide ions is thus analo- tinguished when its container was surrounded by HCl gous to the increased concentration of quinine ions solution. The presence of halides may have caused the needed for maximum emission when the quencher is formation of double or polymolecules of quinine. How- absent. ever, neither conductometric measurements nor freez- On his return to the U.S. Buckingham taught phys- ing-point determinations supported this view. To ob- ics and physical chemistry at Bryn Mawr College from tain any quenching effect by HgCl2, which is only 1893 to 1899 slightly dissociated in solution, a one hundred-fold ex- and then was cess is needed. Thus it is the chloride ion, and not merely briefly affili- a soluble chloride salt, that causes the quenching. Fi- ated with the nally, when Ch.HCl is added to Ch.H2SO4, the fluores- University of cence of the latter is strongly depressed. Thus the effect W isconsin. of halide ions does not depend upon their source. Apparently, he Although Buckingham had examined and elimi- never returned nated various possible causes of the quenching effect, to the quinine he had to admit that he could not explain this effect. topic. He once More than 30 years after he had finished this work, the remarked that following statement appeared in a paper by other work- he had studied ers: “This curious effect of halogen ions remains unex- harmony, not plained (15).” physical chemistry, under In 1928 a double-beam photoelectric fluorimeter Ostwald (17). was used to make a careful study of the quenching of This is a re- the fluorescence of 0.0025M quinine bisulfate solution minder that by increasing concentrations of additives (16). The re- Ostwald, by no sults are summarized in Fig. 6. These confirm that the means a regu- quenching effect is essentially due to the additive an- lar attendant at + ion; Ag is the only cation with appreciable activity. The lectures as a Figure 5. Quenching effect of salts increasing order of quenching efficiency, indicated in on the fluorescence of quinine sulfate Bull. Hist. Chem., VOLUME 27, Number 1 (2002) 61

Table. Quenching Efficiency of Various Ions Quenching Ion: - - 2- - — - - F < NO3 < SO4 < Acetate < Oxalate < Cl < Br < CNS < I Rel. Deformability: 2.5 3.66 3.65 8.7 12.2 18.5

university chemistry student, did manage to learn the Superficial Colour Presented by a Homogenous Liquid viola parts of all of the 83 Haydn string quartets. This Internally Colourless’, “ Philos. Trans. Roy. Soc. Lon- facet of Ostwald’s interests must have appealed to don, 1845, 135, 147-153. Buckingham, who later took miniature scores to sym- 8. D. Brewster, “On a New Phenomenon of Colour in Cer- tain Specimens of Fluorspar,” Report of the Br. Assoc. phony concerts. Adv. Sci., 1838, 10-12. In 1902 Buckingham became a physicist in the U. 9. G. G. Stokes, “On the Change of Refrangibility of Light,” S. Department of Agriculture and then transferred to the Philos. Trans. R. Soc. London, 1852, 142, 463-562. U.S. Bureau of Standards (now the National Institute of 10. G. G. Stokes, “On Certain Reactions of Quinine,” J. Chem. Soc., 1869, 7, 174-181. Standards and Technology). Here he published exten- 11.V. Pierre, “Ueber die durch Fluorescenzhervorgerufene sively on thermodynamics, hydraulics, fluid dynamics, Wärmestrahlungen,” Wien. Akad. Sitzber., 1866, 339- and engineering physics. He retired in 1937 but re- 344; 704-727. mained scientifically active until his death in Washing- 12. E. Hagenbach, “Versuche ueber Fluorescenz,” Ann. ton, DC on April 29, 1940. Phys., 1872, 146, 85-89; 375-405. 13. E. Lommel, “Ueber die Intensität des Fluorescenzlichtes,” Ann. Phys., 1877, 160, 75-96. 14. E. Buckingham, “Ueber einige Fluoreszener REFERENCES AND NOTES scheinungen,” Z. Phys. Chem.(Leipzig), 1894, 14, 129- 148. 1. J. B. Caventou and J. Pelletier, “Nouveau principe amer 15. L. J. Desha, R. E. Sherrill, and L. M. Harrison, “Fluo- acide, crystallisé, contenu dans l’ecorse de la racine rimetry. The Relation between Fluorescence and Hydro- Kahinca,” Ann. Chim. Phys., 1830, 44, 291-296. gen-ion Concentration,” J. Am. Chem. Soc., 1926, 48, 2. P. Rabe, “Zur Kenntnis der China-alkaloide. VIII. Ueber 1493-1500. die Konstitution des Cinchonins,” Ber. Dtsch. Chem. 16. E. Jette and W. West, “Studies on Fuorescence and Pho- Ges., 1908, 41, 62-70. tosensitization in Aqueous Solution. Fluorescence in 3. R. B. Woodward and W. E. Doering, “The Total Synthe- Aqueous Solution,” Proc. Roy. Soc. London, 1928, A121, sis of Quinine,” J. Am. Chem. Soc., 1944, 66, 849. 299-312. 4. G. Stork, Deqiang Niu, A. Fujimoto, E. R. Koft, J. M. 17. F. Perrin, “Role de la viscosité dans les phénomènes de Balkovec, J. R. Tata, and G. R. Dake, “The First fluorescence,” C. R. Hebd. Séances Acad. Sci., Ser. C, Stereoselective Total Synthesis of Quinine,” J. Am. 1924, 178, 2252-2254. Chem. Soc., 2001, 123, 3239-3242. 18. M. D. Hersey, “Edgar Buckingham left his mark at 5. M. J. Baars and G. Patony, “Ultrasensitive Detection of NBS—as an individual and as a leader in thermodynam- Closely Related Angiotension Peptides using Capillary ics,” NBS Standard, 1967 (July), 6. Electrophoresis with Infrared Laser-induced Detection,” Anal. Chem., 1999, 71, 667-671. 6. J. F. W. Herschel, “On a Case of Superficial Colour Pre- ABOUT THE AUTHOR sented by a Homogeneous Liquid Internally Colourless,” Philos. Trans. R. Soc. London, 1845, 135, 143-145. Dr. John T. Stock is Professor Emeritus of Chemistry, 7. J. F. W. Herschel, “On the Epipolic Dispersion of Light, University of Connecticut, Storrs, CT 06269-3060. Being a Supplement to the Paper Entitled ‘On a Case of