Fluorescence Properties of Hydroxy- and Methoxyflavones and the Effect of Shift Reagents

Otto S. Wolfbeis*a, Monowara Begum2, and Hans Geigerb a Institut für Organische Chemie, KF-Universität, A-8010 Graz, Austria b Institut für Chemie der Universität Hohenheim, D-7000 Stuttgart, West Germany Z. Naturforsch. 39b, 231 — 237 (1984); received September 23, 1983 Flavonoids, Fluorescence, Structure Elucidation, Shift Reagents The fluorescence spectra of 42 hydroxy- and methoxyflavones in methanol solution have been investigated. The following findings are considered to be useful in structure elucidation and identification of flavonoids: (a) The maxima of the absorption and fluorescence bands give, in most cases, a unique combination; (b) flavones exhibit exceptionally large Stokes’ shifts (6,800 to 10,000 cm-1); (c) most flavones fluoresce blue, but flavonols fluoresce yellow or green; the fluorescence of flavonols consists frequently of two bands; (d) fluorescence is zero or very weak for 5-hydroxyflavones; (e) 5-hydroxyflavones become increasingly fluorescent with in increasing number of oxygen functions being present in the molecule; a 3-hydroxy group has a particular beneficial effect; (f) hydroxyflavones fluoresce less intense than the corresponding methoxy­ flavones; (g) fluorescence intensity is distinctly higher in polar protic than in apolar solvents. The effects of three groups of shift reagents on the spectra has also been investigated. The first group involves water, 10% and 50% sulphuric acid. The second group consists of basic reagents (sodium acetate, sodium hydrogen carbonate and sodium carbonate), and the third group of complexing reagents such as aluminum trichloride, borax and magnesium sulphate. The following generalisations may be made: (a) Water causes a bathochromic shift in emission with 7-hydroxy- flavones, but a hypsochromic shift with flavonols; (b) 50% sulphuric acid is able to protonate (and thus to longware-shift the emission maximum) of all flavones except for the 3-, 5-, and 8 -hydroxy isomers; (c) sodium acetate and hydrogen carbonate cause characteristic shifts with 7-hydroxy- flavones and, occasionally, with 4'-hydroxyflavones lacking a 7-hydroxy group; (d) aluminum trichloride produces a longware shift and an enormous increae in fluorescence intensity with flavonols; (e) borax can be used to detect sensitively the presence of a 3',4'-dihydroxy group.

Introduction which may be used to identify flavones. The aim of Ultraviolet spectrometry without and with added the present work is to provide a composition of the shift reagents is one of the most widely used methods solution spectra of 42 flavone aglycons in the pre­ for the identification of flavonoids [1]. In comparison sence and absence of various shift reagents. with U.V. methods, fluorimetry is a more sensitive means for the detection and quantitation of fluores­ cent compounds. In the most simple case the fluorescence band of a molecule is the mirror image of its longest-wave absorption band, shifted to longer wavelengths. So far fluorimetry would provide no more information than does absorptiometry. In addi­ The effects of the following shift reagents, which tion to previous work on the fluorescence of are readily available in every laboratory, were inves­ flavonoids [2] and of 6 ,8- and 2'-hydroxyflavones [3] tigated: on tic plates and in the solid state [4] some recent (1) Water. Since most of the used shift reagent solu­ studies on the fluorescence properties of selected tions are aqueous, we had to check the influence hydroxyflavones have revealed [5—9] that these of water. It turned out that water itself is a useful molecules may form exciplexes, undergo tautomeric reagent; changes or photodissociation. These processes are (2) 50% sulphuric acid and, less suitable, 10% sul­ highly dependent upon the solvent and its acidity and phuric acid; give rise to unusually longwave emission bands, (3) sodium acetate; (4) sodium hydrogen carbonate; (5) sodium carbonate; * Reprint requests to Dr. O. Wolfbeis. (6) sodium hydroxide; 0340-5087/84/0200-0231/S 01.00/0 (7) aluminum trichloride; 232 O. S. W olfbeis et al. ■ Fluorescence Properties of Hydroxy- and Methoxyflavones

(8) borax (Na 2B40 7), and oxygen functions being present, the intensity begins (9) magnesium acetate/triethylamine. to increase. A 3-hydroxy group has a particularly Neu’s reagent (diphenyl boric acid /3-aminoethyl- beneficial effect (entries 30, 34 and 35 in Table II). ester), which is such a useful shift reagent for 5-Methoxyflavones are strongly fluorescent. Simi­ flavonoids absorbed on cellulose thin layers [3, 4], is larly, all other methoxyflavones are more strongly of little use for solution measurements. On the one fluorescent than the respective hydroxyflavones side it complexes only slowly to give weakly fluores­ (Tables II, III and IV). cent products, on the other side it exhibits a weak Addition of three volumes of water to one volume fluorescence itself. of the stock solution results in spectral shifts, changes in intensity and broadening of the bands. Batho- Results chromic shifts are observed with 2'- and 3'-hydroxy- With the exception of the 5-hydroxy derivatives all flavones and, less so, with their methyl esthers. In flavones are fluorescent in methanol solution. the case of flavonols, addition of water causes hypso- Fluorescence is distinctly lower in acetonitrile and chromic shifts of the longwave band, but opposite drops even more in cyclohexane. The relative inten­ shifts of the shortwave band. Compounds with a free sities are given qualitatively in the Tables. To allow a 7-hydroxy group frequently exhibit an additional rough estimation of the quantum yields we have band at around 520 nm when water is added. Minor compared our intensity scale with some measured shifts produced with other hydroxyflavones (Table quantum yields (Table I). The threshold of percep­ II) cannot be explained at present, but are neverthe­ tion of a well-adapted eye is in the intensity range 2 less useful in characterising and identifying specific to 3. flavones. Flavones with methoxy substituents but without Addition of 50% sulphuric acid results in longwave hydroxy substituents show only one single fluores­ shifts of 40 to 110 nm with all flavones lacking a cence band of rather high intensity. Those having 3-, 5- or 8-hydroxy group due to protonation. 5-Hy- one or more hydroxy groups (in particular in 3- or 7- droxyflavones with a 3'-methoxy group are, how­ position) may exhibit two fluorescence bands or un- ever, an exception in giving a weak green emission symmetric shoulders along with the main band. Ex­ too. cept for the flavonols all investigated hydroxy- and The effect of basic shift reagents has been investi­ methoxyflavones have their fluorescence maxima in gated with all compounds bearing hydroxy groups the 403 to 480 nm range. (Table III). We notice that extremely pure reagents 3-Hydroxyflavones (“flavonols”) show green or should be used to obtain reproducible results. In par­ yellow emissions with maxima at above 500 nm and ticular, they should be checked not to contain frequently a second, rather weak band in the blue aluminum, magnesium and other complex forming part of the spectrum [5, 6 , 9], ions. There are drastic differences in fluorescence inten­ Addition of one part of NaOAc solution to three sity evident. Generally, 5-hydroxyflavones fluoresce ml of the stock causes two effects in emission: The very weak, if at all. With an increasing number of one results from changing to an aqueous solvent sys­ tem and is similar to that produced by water. The Table I. Fluorescence quantum yields (0f) of some oxy- other results from the basicity of the reagent and is flavones in water at room temperature in comparison with very distinct for all hydroxyflavones having either a the relative intensity scale used in the Tables. 7- or 4'-hydroxy group (Table III). With 7-hydroxy- Flavone 0f Relative intensity Ref. flavones the maxima are shifted to above 500 nm, (scale 1 to 6) except for 7-hydroxy-3',4'-dimethoxyflavone and 3-OH 0.005 3a [9] some of the tetra- and pentahvdroxyflavones, with 7-OH 0.004 3a [7] which only shoulders at above 500 nm may be pro­ 4 -O H 0.02 4 [8 ] 4'-OM e 0.95 6 [8 ] duced. Unfortunately, the reagent is not specific for 7-hydroxyflavones, since 4'-hydroxvflavones without a Fluorescence intensities in the case of low quantum yields a 7-hydroxy group may also be affected. can not be estimated visually with accuracy. Therefore deviations of ± 1 in the relative intensity scale range 1 to With N aH C 0 3 reagent again the water and base 2 are possible. effects have to be discerned. Unlike NaOAc it pro- O. S. Wolfbeis et al. • Fluorescence Properties of Hydroxy- and Methoxyflavones 233

Table II. Fluorescence maxima (nm) of flavones in methanol and the effect of water and acidic shift reagents thereon. Intensities — given in brackets — range from 6 (very high) to 1 (very low) (see Table I).

No. Flavone Fluorescencemaximum OH OMe Methanol +Water + 10% h 2s o 4 +50% H 2SO

1 3 _ 405, 531 (3) 412, 514 (3) 410, 528 (3) 412, 529 (3) 2 5 - a a - a - a - 3 - 5 459 (4) 473 (5) 464, 525 (4) 535 (3) 4 6 - 455 (4) 461 (4) 462 (4) 510 (3) 5 - 6 444 (5) 459 (6) 498 (5) 498 (4) 6 7 - 419, 543 (3) 540 (3) 432, 543 (4) 428, 542 (3) 7 - 7 404 (4) 414 (4) 440 (4) 440 (4) __C 8 8 - 471 ( 1) 475 (2) 480 ( 1) 9 - 8 475 (2) 495 (3) 485 (2) 468, 554 (2) 10 2' - 427 (2) 452 (3) 429, 520 (sh) 427, 514 (2) 11 - 2' 428 (3) 452 (4) 435, 509 (3) 516 (3) 12 3' - 428 ( 1) 452 (2) 450 (2) 457 (2) 13 - 3' 438 (2) 466 (3) 440, 460 (sh) (3) 547 (2) 540 (sh) 14 4' - 421 (4) 428 (4) 425 (4) 466 (3) 15 - 4' 412 (6) 424 (6) 462 (5) 462 (4) 16d 5 7 460 ( 1) 460 (1) 520 (2) 520 (2) 17 7 5 460 (3) 471, 520 (sh) 509 (br) (3) 520 (4) 18 - 5, 7 460 (4) 478 (4) 430 (sh), 513 (4) 513 (4) 19 7, 4' - 413 (5) 423, 500 (sh) 428, 460 (sh) (3) 457 (4) 520 20 - 7, 4' 406 (6) 416 (5) 474 (5) 473 (6) 21 7 4' 405, 530 (5) 415, 525 (4) 446, 470, (3) 455 (4) 518 22 3', 4' - 466 (3) 460 (3) 455 (3) 475 (2) 23 - 3', 4' 460 (4) 475 (4) 463 (4) 532 (3) 24e 3, 5, 7 - 421, 530 (1) 420, 514 (1) 428, 523 (1) 440, 523 (sh) _C _C 25f 5, 7, 4' — 434 (1) 440, 473b (1) 500 (sh) 26« 5, 7 4' -- -- 27 - 5, 7, 4' 430 (4) 458 (3) 488 (2) 487 (2) 28 7, 3', 4' - 460 (2) 480 (very br.) 470, 520 (sh) (2) 510 (2) 29 7 3', 4' 440 (4) 458 (3) 448, 510 (sh) 518 (2) 30h 3, 5, 7, 4' - 540 (2) 427 (sh) (2) 432, 520 (1) 432 (sh), 497 (2) 462 (sh), 520 31* 5, 7, 3', 4' -- 440, 513 (sh) 435, 510 (2) 438 (2) 32 3, 5, 7 4' 535 (2) 530 (2) - 500 ( 1) 33 5, 7 3, 4' - 435, 510 (2) 435 (2) 435, 470 (sh) (2) 34J 3, 5, 7, — 435, 505, (2) 440(sh), 501 431 (sh) (2) 431, 560 (2) 2', 4' 550 (sh) 495, 560 35k 3, 5, 7, — 430, 480, (2) 460, 523 (2) 433, 532 (2) 433, 532 (2) 3', 4' 533 36 — 3, 5, 7, 445 (3) 457 (2) 450 (2) 528 (2) 3', 4' 37 5, 7, 2', - 465, 520 (br) 438, 510 ( 1) 435, (1) - 4', 5' 500-560 (br) 38 — 5, 7, 2', 475 (4) 490 (4) 490 (3) 557 (3) 4', 5' __C __C 39‘ 5, 7, 3', 4', 5' - 465 (2) 465 (2) 40m 3, 5, 7, 4' 3' 485, 550 (sh) (3) 491 (3) - 485 (2) 41" 5, 7, 4' 3', 5' 463 (2) 463 (2) - 463, 520 (br) (2) 42° 3, 5, 7, 492 (2) 494, (2) 486 (2) 3', 4', 5' 550 (sh)

a No fluorescence detectable; b compound appears to decompose;c fluorescence is quenched; d tectochrysin;e galangin; f apigenin; 8 acacetin; h kaempferol; 1 kaepferid; j morin; k quercetin; 1 tricetin; m isorhamnetin; n tricin; ° myrcetin. 234 O. S. Wolfbeis et al. • Fluorescence Properties of Hydroxy- and Methoxyflavones

Table III. Effect of basic shift reagents upon the fluorescence maxima of hydroxyflavones, and intensities of the fluores­ cence bands (in brackets).

No. Flavone Fluorescence maximum (nm) OH OMe + NaO A c + N a H C 0 3 + Na 2C 0 3 +N aO H

1 3 - 410, 514 (3) 430 (sh), 516 (4) 430, 520 (3) 520a (3) 2 5 _b 4 6 - 466 (4) 464 (3) 482 (2) 6 7 - 425, 528 (3) 434, 517 (3) 530 (3) 530 (3) 8 8 - 469 ( 1) 470 (1) 474 ( 1) 508 ( 1) _b 10 2' - 432 (3) 458 (2) 463 (2) 12 3' - 455 (2) 455 (2) 460 (2) 445, 510 ( 1) 14 4' - 425 (4) 424 (4) - 5 0 0 (4) 511 (3) 16 5 7 460 ( 1) 460 ( 1) 460 (1) - 17 7 5 4 6 0 -5 2 0 (4) 525 (4) 527 (4) - 19 7, 4' - 514 (4) 483 (4) 480 (4) 480 (4) 21 7 4' 520 (5) 516 (5) 516 (5) 516 (4) 22 3', 4' - 460a (3) 475a (3) 516 (2) 515 (2) 24 3, 5, 7 - 480 (sh), 553 (1) 492 (sh), 559 (1) 497 (sh), 570 (1) - 25 5, 7, 4' - 442, 503 ( 1) 480 ( 1) 490 (1) - 26 5, 7 4' 434, 520 ( 1) 459 0 ) 470 (sh), 511 (1) - 28 7, 3', 4' - 516 (3) 516 (3) 516 (3) - 29 7 3'., 4' 450, 510 (sh) (4) 520 (3) 520 (3) - 30 3, 5, 7, 4' - 460 (sh), 530 (2) 491, 550 (2) 551 (2) - 31 5, 7, 3' , 4' - 511 ( 1) 460, 511 (sh) (2) 520 (2) - 32 3, 5, 7 4' 533 (3) 533 (3) - - 33 5, 7 3, 4' 430, 510 (sh) (2) 430, 509 (2) __C - 34 3, 5, 7, 2 ', 4' - 436, 557 (2) 516 (sh), 560 (4) 560 (4) - 35 3, 5, 7, 3', 4' - 460, 543 (2) 510 (3) 540 (3) - 37 5, 7, 2' , 4' , 5' - 436, 520 (2) 435, 460 (2) 460 (sh), 506 (2) - 520 (sh) 39 5, 7, 3' , 4' , 5' - 465, 540 (sh) (2) 538 (3) -- 40 3, 5, 7, 4' 3' 460 (sh), 540 (3) 555 (3) -- 41 5, 7, 4' 3'., 5' 463 (sh), 530 (2) 533 (3) -- 42 3, 5, 7, 3', 4', 5 '- 492, 584 (2) 552a (2) - -

J Decomposes with tim e;b quenching; c A 480 nm-band was observed, but this arises most likely from the Mg-complex. Magnesium(II)ion appears to be present even in high quality sodium carbonate.

duces the anion fluorescence of all 7-hydroxy- Aluminum trichloride is a frequently used chelat­ flavones, but like NaOAc it may also indicate a 4'- ing shift reagent for the identification of 3- or 5-hy­ hydroxy group. droxyflavones by u.v. spectrometry. We have Along with the shifts that are already produced studied the effect of this reagent with all flavones with NaHC03, sodium carbonate reagent induces containing at least one hydroxy group (Table IV). It shifts for all 4'-hydroxyflavones (weak fluorescences is able to enhance the fluorescence intensity of all maximising at around 500 nm), and 3',4'-dihydroxy- flavonols by a factor of sometimes more than flavones. If a 5-hydroxy or a 3'-hydroxy group is the hundred, and gives frequently rise to a new, single only dissociable group being present, the effect of emission band even in cases where the stock solution Na2C 0 3 remains poor. spectrum exhibits a complicated pattern. Also af­ With sodium hydroxide a decrease in fluorescence fected are 5-hydroxyflavones, which give longwave intensity was observed in most instances and shift green emissions, but their intensity increases by far were not uniform. With polyhvdroxyflavones mix­ not as much. tures of mono- and polyanions may be formed, a fact With 3'.4'-dihydroxyflavones aluminum tri­ that can result in the appearance of several, mostly chloride forms also strongly fluorescent complexes, broad bands. Thus, only mono- and dihydroxy- whereas compounds with isolated 3'- or 4'-hydroxy flavones were investigated. groups remain unaffected. O. S. W olfbeis et al. • Fluorescence Properties of Hydroxy- and Methoxyflavones 235

With borax as a shift reagent (Table IV) the water, pleteness, the data obtained with the latter two rea­ base and complexing effect have to be distinguished. gents are included in the Tables. The former two can be recognised by looking at the effect of water and sodium carbonate. Only with Discussion 3',4'-dihydroxyflavones additional fluorescences are The fact, that 5-hydroxyflavones are practically observed, that are thought to result from complexa- non-fluorescent provides a simple means to recog­ tion. Thus, this kind of functional group may be nise them. The fluorescence maxima as given in identified. Table II could only be obtained by scanning the We have also studied the effects of two other rea­ spectra with a very sensitive photomultiplier tube gents, which, however, proved to be less useful. 10 % and at bandpasses of above 15 nm at both the excita­ sulphuric acid can produce shifts by more than 35 nm tion and emission monochromator. when added to flavones without a free hydroxy group Our fluorescence intensity scale as presented in (Table II). Unfortunately there are too many excep­ Table I covers a range of approx. five exponential tions to make a generalisation, so that this reagent is units. Clearly, the exact value of the fluorescence thought to be of limited diagnostic value. quantum yield would be the physically meaningful Aqueous magnesium sulphate alone did not pro­ measure for the intensity, but unlike the extinction duce an effect in addition to the water effect. In al­ coefficient, a quantum yield can be determined only kaline solutions (prepared by adding triethylamine by tedious measurements, which are far beyond and filtration) complexation occurs with flavonols routine. To allow a rough estimate of the magnitude and 3',4'-dihydroxyflavones, functions that can be of the quantum yield, some previously determined recognised quite easily by other methods. For com­ values are given in Table I. If our spectra are to be

Table IV. The effect of complexing shift reagents upon the fluorescence maxima of hydroxyflavones, and intensities of the fluorescence bands (in brackets).

No. Flavone Fluorescence maximum (nm) OH OMe +AICI3 + Borax +MgS0 4/E t3N

1 3 - 466 (6) 410, 524 (4) 484 (4) 2 5 - 580 (1 ) -- 4 6 - 467 (4) 472 (3) 460 (2) 6 7 - 432, 534 (3) 528 (3) 528 (3) 8 8 - 470 (1 ) 510 (2) 464 (2) 10 2' - 450 (3) 4 8 0 -4 9 0 (3) 453, 507 (sh) (4) 12 3' - 432 (2) 425 (2) 430 (2) 14 4' - 441, 500 (sh) (3) 439 (4) 442, 500 (sh) (3) 16 5 7 547 (3) 460 (2) 460 (2) 17 7 5 460 (4) 526 (4) 520 (4) 19 7, 4' - 432 (5) 480 (5) 480 (4) 21 7 4' 435, 520 (sh) (5) 519 (5) 515 (5) 22 3', 4' - 539 (3) 520 (4) 509 (3) 24 3, 5, 7 - 487 (6) 534 (3) 533 (3) 25 5, 7, 4' - 523 (2) 450 (2) 488 (1 ) 26 5, 7 4' 522 (2) 468 (sh), 532 (1) 510 (br) (2) 28 7, 3', 4' - 550 (3) 500 (4) 505 (4) 29 7 3', 4' 444 (3) 445 (sh), 520 (3) 520 (3) 30 3, 5, 7, 4' - 483 (5) 534 (3) 520 (3) 31 5, 7, 3', 4' - 540 (4) 436, 517 (2) 520 (3) 32 3, 5, 7 4' 520 (4) 533 (3) - 33 5, 7 3, 4' 440, 523 (3) 431, 510 (3) 430 (sh), 485 (2) 34 3, 5, 7, 2', 4' - 494 (5) 549 (4) 525 (4) 35 3, 5, 7, 3', 4' - 537 (5) 535 (4) 537 (4) 37 5, 7, 2', 4', 5' - 564 (3) 533 (2) 508 (2) 39 5, 7, 3', 4', 5' - 514 (4) 526 (3) - 40 3, 5, 7, 4' 3' 489 (5) 533 (3) - 41 5, 7, 4' 3', 5' 520 (3) 533 (3) - 42 3, 5, 7, 3', 4', 5' - 540 (4) 531 (3) - 236 O. S. Wolfbeis et al. ■ Fluorescence Properties of Hydroxy- and Methoxyflavones compared with spectra obtained with another in­ The action of 10% sulphuric acid is intermediate strumentation we suggest to calibrate it with a few of between water and 50% acid. The reagent is able to the more readily available compounds. protonate, possibly only in the excited state, most of We assume that the lack of fluorescence of 5-hy- the methoxyflavones lacking a free hydroxy group, droxyflavones is the result of a diabatic excited state but the reagent is far away from being specific for proton transfer, since 5-methoxyflavones are highly methoxyflavones. fluorescent. In a study on some polyhydroxyflavones When comparing the sodium acetate with the wa­ Kuhn and Low [10] have pointed out, that those hav­ ter effect one notes that practically all 7-hydroxy­ ing a 5-hydroxy group can be distinguished from flavones suffer a shift to above 500 nm. Like in ab­ their isomers having a 5-methoxy group by observing sorption spectrometry, the reagent is therefore suit­ their fluorescence in acetic anhydride solution. Only able to recognise 7-hydroxyflavones and may give, in the latter are fluorescent, but addition of B 20 3 make the absence of this group, a hint for the presence of a the 5-hydroxyflavones fluorescent too. 4'-hydroxy group. The effect of NaHC0 3 is similar to Generally it is found that methoxyflavones are that of sodium acetate. Due to its more basic charac­ stronger fluorescent than the respective hydroxy- ter it is able to produce the anion fluorescence of all flavones. In view of the results on the excited state 7-hydroxyflavones and most of the 4'-hydroxy- photophysics of flavones, which are known to suffer flavones that lack a 7-hydroxy group. drastic changes in their pKa values after photoexcita­ The effects of sodium carbonate and sodium tion [7—9] we assume that the low fluorescence in­ hydroxide may be used to indentify unknown tensities of hydroxyflavones result from incomplete flavonoids by comparison of the obtained spectra excited state proton transfer reactions, thus giving with the data presented in this work, but the reagents rise to more efficient non-radiative deactivation. are not suitable to elucidate the positions of hydroxy The appearance of two emission bands for some groups. flavonols can, by analogy to the behaviour of the The low fluorescence quantum yields of flavonoids parent compound [5, 6 , 9] be interpreted in terms of in alkaline solutions are in significant contrast to the an excited state intramolecular proton transfer. The behaviour of hydroxycoumarins, whose fluores­ blue band is assigned to the non-tautomerised cences are known to be highest in alkaline solution molecule, whilst the green of yellow ones arise from [13]. the excited state tautomer. Aluminum trichloride is a superb reagent for the The effect of added water can be interpreted with identification of flavonols. Not only it produces a the help of results obtained with model compounds: sharp and single band, but also increases the intensi­ The longwave emissions from 7-hydroxyflavones are ty drastically. The increase by a factor of ten or more from the corresponding anions, which are formed may be used to distinguish flavonols from 5-hy- through excited state photodissociation even in droxyflavones, the intensity of whose A1C1 3 complex weakly acidic solution [7], The hypsochromic shift of is much lower. 3',4'-Dihydroxyflavones interfere, the flavonol tautomer band after addition of water is but the presence of such a function can easily be a solvent effect. recognised with borax, which is practically specific 50% sulphuric acid is strong enough to protonate therefore. almost all flavones without a 5-hydroxy group. Prob­ As a result, the following four reagents are con­ ably, those with a 8-hydroxy group are also not pro- sidered to be useful in fluorimetric structure elucida­ tonated (only one example). Thus, a shift induced by tion and identification of oxyflavones: 50% sulphuric acid to near or above 500 nm indi­ 1)50% sulphuric acid (either 3-, 5-, or 8-hydroxy- cates the absence of a 5-hydroxy group, except for flavone, or not); flavonols, which themselves fluoresce greenish. The 2) NaHCO? reagent (either 7- or 4'-hydroxyflavone lack of protonation by 50% acid seems to be the or not); result of the low basicity of 5-hydroxyflavones, and 3) A1C13 reagent (flavonol or not; 5-hydroxy- and some of the flavonols. It is known, that 5-hy­ 3',4'-dihydroxyflavones can interfere); droxyflavones and, less so. flavonols possess higher 4) borax (3',4'-dihydroxyflavone or not; specific, if dissociation constants and lower protonation con­ no 7-hydroxy group being present; other ortho- stants than the residual hydroxyflavones [ 10 , 1 1 ], dihydroxvflavones not investigated). O. S. W olfbeis et al. ■ Fluorescence Properties of Hydroxy- and Methoxyflavones 237

Experimental reagent solution were added and the effect upon the Samples. — All investigated compounds had been spectrum observed. isolated from natural sources or were synthesized by When adding either sodium hydrogen carbonate or magnesium sulphate together with triethylamine, known methods. They were purified by chromato­ a precipitate was formed, which had to be removed graphy and/or repeated crystallisation and did not by filtration. When using water as a shift reagent, the contain tic visible impurities. As far as controlled, dilution was one ml of the stock with three ml of the excitation spectra were in reasonable agreement water. with the absorption spectra. Instrumentation. — The spectra are uncorrected Reagents. — Methanol (Merck, for fluorescence and were recorded in rectangular cells (lx l cm). spectroscopy) was used generally as solvent. Water The excitation beam was vertically to the emission was distilled, first from potassium permanganate, beam. then twice from pyrex glass. 50% sulphuric acid was Excitation was done at one of the mercury lines at prepared from 50 g sulphuric acid (96%) and 50 g 313 and 366 nm, since most of the commercial HPLC water under cooling. Sodium acetate reagent: 5 g instrumentation is equipped with mercury lamps as NaOAc • 3 H 20 in 100 ml water. Sodium hydrogen light sources in the detection unit. The 313 nm line carbonate reagent: 1 g NaHC0 3 in 100 ml water. was choosen to excite the monhydroxyflavones, the Sodium carbonate: 1 g Na 2C 0 3 • 10 H20 in 100 ml 366 nm line to excite the polyhydroxyflavones. In water. Sodium hydroxide: A solution of 4 g NaOH some cases excitation was performed to at 340 to (solid) in 100 ml water. Aluminum trichloride: 350 nm (near the absorption maximum) in order to Anhydrous A1C1 3 (1 g) dissolved under cooling in produce a measurable fluorescence. The reproduci­ 100 ml methanol. Borax: 1 g Na 2B40 7 • 10 H 20 bility of the emission maxima is within ± 2 nm. in 100 ml water. Magnesium sulphate: 1 g M gS0 4 • 7 H20 in 100 ml water. Triethylamine: Commercial triethylamine was distilled over sodium wire. This work was supported by the “Fonds zur För­ Experimental. — Approx. 10-5 to 10 -6 M stock derung der wiss. Forschung”, project no. 4432, solutions of the flavonoids were prepared in which is gratefully acknowledged. M. B. thanks the methanol. Three ml of the stock were transferred Ministerium für Wissenschaft und Forschung for a into the quartz cell and the fluorescence spectra of stipend, and H. G. the “Fonds der Chemischen In­ this solution were measured. Then 1 ml of the shift dustrie” for financial support.

[1] T. J. Mabry, K. R. Marham, and M. B. Thomas, “The [9] O. S. Wolfbeis, A. Knierzinger, and R. Schipfer, J. Systematic Identification of Flavonoids”, Springer Photochem. 21, 67 (1983); on flavonol. Verlag, Berlin 1979. [10] R. Kuhn and I. Löw, Ber. Dtsch. Chem. Ges. 77, 211 [2] M. Kopach, S. Kopach, and W. Klechek, Zhurn. Org. (1944). Khim. 16, 1721 (1980); engl. ed. p. 1467. [11] a) The pKa value of 5-hydroxyflavone is as high as [3] H. Homberg and H. Geiger, Phytochemistry 19, 2443 11.56: N. A. Tyukavkina andN. N. Pogodaeva, Khim. (1980). Prir. Soedin. 1972, 173; engl. ed. p. 178; [4] H. Geiger and H. Homberg, Z. Naturforsch. 38b, 253 b) The pKa value of the conjugate acid is —3.07: N. N. (1983). Pogodaeva and N. A. Tyukavkina, Khim. Prir. [5] P. K. Sengupta and M. Kasha, Chem. Phys. Lett. 68 , Soedin. 1973, 25; engl. ed. p. 22. 382 (1979); on flavonol. [12] a) The pKa value of flavonol is 9.6: N. A. Tyukavkina [6] G. J. W oolfe and P. J. Thistlethwaite, J. Am. Chem. and N. N. Pogodaeva, Khim. Prir. Soedin. 1971, 11; Soc. 103, 6916 (1981); on flavonol. engl. ed. p. 8 ; [7] R. Schipfer, O. S. Wolfbeis, and A. Knierzinger, J. b) The pKa value of the conjugate acid is —2.88: ref. Chem. Soc. Perkin II 1981, 1443; on 7-oxyflavones. [lib ]. [8 ] O. S. Wolfbeis and R. Schipfer. Ber. Bunsenges. [13] D. G. Crosby and R. V. Berthold, Anal. Biochem. 4, Phys. Chem. 86, 273 (1982); on 4'-oxyflavones. 349 (1962).