MARCH. 1953
FLUORESCENCE TITRATION JACK DE MENT De Ment Laboratories, Portland, Oregon
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titration of opaque, highly colored, and similar solutions has always been a problem in analytical chemistry. Such a problem is often solved by the use of fluorescent indicators. Fluorescent indicators are substances which show definite changes in fluoresence with change in pH, provided an end point occurs. Fluorescent indicators may change from one fluorescent color to another, or the indicators may serve because of the appearance or disappearance of fluorescence at certain pH values. Fluorescent iudicators often have excellent and sharp end points. As such indicators are usually organic compounds such as dyestuffs and alkaloids, the converse approach permits in many instances the fluorochemical assay of the indicator itself. This can be especially valuable in medical work where, for example, quinine can be determined (10, 16). Thus, the iutensity of the fluorescence of quinine in dilute sulfuric acid at room temperature and pH 2 bears a linear relationship to the concentration (5). In fact, quinine is often used as a standard in the more elaborate fluorometric methods which involve instruments for this purpose. Fluorescence titrations are best carried out in semidarkness or in total darkness. A long-wave-length ultraviolet lamp, equipped with a filter, replaces the usual source of light. A few particles of insoluble
fluorescent material are dropped onto the surface of the liquid in the buret, in order to follow the meniscus. Or, a few particles of fluorescein or quinine on the surfaces of basic and acidic solutions, respectively, can be used. There are many fluorescent indicators available and the techniques can often be varied to fit the problem (cf. 9, 5). The organic indicators are of most general value, whereas in special problems inorganic indicators are frequently helpful. Uranyl salts have been proposed and used from time to time, but the influence of various ions upon the fluorescence of uranyl solutions is usually too great to permit accurate work. Considerable study has been done in this direction on the halogen ions and others (52). In place of methyl orange or methyl red in bromidebromate titrations fluorescein has been found satisfactory, as its fluorescenceis not so likely to be destroyed by free bromine (17). If a small amount of freshly prepared 5-aminosalicylic acid solution is added to sodium hydroxide solution and this titrated under ultraviolet light, the fluorescence disappears as soon as all of the base has been neutralized. The results obtained agree with those for methyl orange (21). Fluorescent indicators may exhibit shortcomings
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JOURNAL O F CHEMICAL EDUCATION
common to other organic indicators. Certain fluorescent substances react with heavy metal ions, d i s h ing their effectiveness in titration. Nevertheless, this unique class of indicators is often profitably relied upon in specialized or unusual problems. In fluorescence titrimetry the buret and other glassware, especially the receiving vessels, should be nonfluorescent, so that clear results are obtained. The glassware stock of the laboratory is first examined under ultraviolet light and those articles which do fluoresce are not employed. APPLICATIONS
CATALOGUE OF INDICATORS
In the following list of some 60 fluorescent organic indicators a division is made according to the approximate pH range covered. pH0Dt.2
BolzqAavine: yellow fluorescent at pH 0.3,changing to green a t oH 1.7 (1.9). 6-~io~y~hlhalimide: blue fluorescent a t pH 0, changing to green fluoresoent a t pH 2.4 (19, 18). Eoszn: colorless a t pH 0 and green a t pH 3.0(6,18,67). Eosin G: similar to above, having the same transition points, except that the fluorescence is yellowish a t pH 3.0( i d ) . Eosin YS: yellow colored a t pH 0 and fluorescent a t pH 3.0
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(7,
Many branches of chemistry benefit by adoption of fluorescent indicators and the ultraviolet lamp. The lamp itself is a valuable tool which can be applied to many other problems, aside from those of volumetric analysis (cf. 9, 10, 9, 18, 99). The opaque, turbid, or deeply colored liquids which are titrated under ultraviolet light should not exhibit an intense fluorescence of their own, as this interferes with the fluorescence of the titration. In some instances, the addition of a quencher, an agent which reduces or destroys the fluorescence, facilitates titration. The best quenchers are usually compounds with nitro groups or with several halogen atoms. In particular, the food industries and the fermentation arts find fluorescence titration valnable. Examples include colored wines and other liquors, fruit juices, dark vinegars, and plant extracts. In pharmacy, colored tinctures and extracts yield to the method. In agriculture, soil extracts and like samples can be titrated. Fluorescence titrations can be employed to advantage in microchemistry and ultramicrochemistry where exceedingly small amounts of liquid are handled, often with the assistance of the microscope. A fluorescent substance also helps in following a liquid in a capillary tube or in a microburet, and is useful when colors or color changes cannot be easily discriminated or are apt to be deceiving. The determination of the ionization constants of complex organic compounds has been claimed feasible similar titration (297 ' 4 ) . The measurement of ionization constants for such substances as quinine sulfate, methoxyquinoline, and 8-naphthol has been found to be in agreement with values obtained by other techniques. The fact that isomers of various oreanic com~ounds vary in their fluorescent qualities with respect to pH provides a new and quick method of distinguishing such substances. The same is true of complicated organic molecules which vary slightly in composition or structure. In such methods fluorescence intensity is plotted against pH. Thus, the a- and p-naphthylamines vary; also the many naphtholsnlfonic. acids, and the naphthionic acids (9). The various eosins, for example, show different fluorescent responses with changes in pH.
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Ervlhrosin: oranee nt DH 0 and fluoresceut at oH 3.6 ( 7 )
9, 6-Tetramethyldiaminozanthae: green fluorescent a t pH 1.2, blue fluorescent at pH 3.4 (19). at-4
Chmotropic acid: colorless at pH 3.5,blue fluorescent a t pH 4.5 (28). Eosin (C.I. 68): colorless at pH 2.5,green fluorescent at pH 4.5 (7; cf. previous data for eosin). Fluorescein (C.I. 766): colorless a t pH 4, green fluorescent at pH 4.5 ($4, d ) . Magdala Red: purple colored at pH 3.0,fluorerrcent s t pH 4.0 (91). or-Naphthvlamine: colorless at DH 3.4,blue fluorescent a t DH 4.8 ( u j . PNaphthylamine: colorless a t pH 2.8,violet fluorescent a t pH 4.4 (18). This indicator bas been described as having a second end point, colorless a t pH 3.4 and blue fluoresoent a t pH 4.8 119 - ,. - - , -18) Phlozine (C.I. 774): colorless a t pH 3.4 and bright yellow fluoreseent a t pH 5.0. Some brands vary in behavior. Salicylic acid: colorless at pH 2.5,blue fluoreseent s t pH 3.5. \
pH 4 to 6
Acridine (C.Z. 788): green fluorescent a t pH 4.9,violet at pH 5.1. The value 5.0has $80 been given (83). DichlorgRuorescein: usually colorless a t pH 4.0, and bright green fluorescent a t pH 6.0 (18). The value 6.6 has also been given instead of 6.0 ( 8 ) . 3, 6-Diozyzanthone: colorless a t pH 5.4,blue-violet fluorescent a t pH 7.6 (19). Eosin (C.Z. 768) and Xylene Cyanol FF (C.I. 716), in ethanol and mbutanol: colorless at pH 4.0,green fluoresoent a t pH 5.0 (86). Endthrosin (C.I. 772): colorless at pH 4.0,light fluorescent a t pH 4.5 (18). The fluorescent color a t PH 4.0 has also been stated as green. &,M:t?ylegeuletin: colorless at pH 4.0,blue fluorescent a t pH 0 . X (18, 1Yj.
Neville-Winther acid: colorless a t pH 6.0,blue fluorescent at ..u ".., R e s o n ~ f i : yellow fluorescent a t pH 4.4 and a weak orange at pH 6.4(19). This indicator sometimes is eolarless a t pH 4.4and a stronger orange s t pH 6.4,apparently depending upon the praduct. Qvininie acid: yellow a t pH 4.0,blue fluoreseent a t pH 5.0 (SO).
Quinine (first end point): blue fluorescent a t pH 5.0,violet fluorescent s t pH 6.1 ( i d , 11, 86, 14). pH 6 to 8
Acid R Phosphine: claimed to he useful in the range pH 6.07.0.
MARCH, 1953 Brilliant Diazo1 Yellmu: colorless a t pH 6.5, violet fluorescent s t pH 7.5 (1s). Cleves acid: colorless a t pH 6.5, green fluorescent at pH 7.5 (18). Cmmaric aeid: colorless a t pH 7.2, green fluorescent a t pH 9.0 (19). 3, 6-Dioz?,phthalic dinitrile: blue fluorescent a t pH 5.8, green fluorescent at pH 8.2 (13). Magnesium 8-hydrozyquinolina&: colorless a t pH 6.5, golden colored fluorescence a t pH 7.5 (16). 8-Metholumbellifmne: colorless at DH 7.0. h e blue fluoresce& a t 7.5 ((I,83, $3). 1-Naphthol-4-sulfonic aeid: colorless at pH 6.0, bluc fluorescent at pH 6.5 (12). Orcinaunne: colorless a t pH 6.5, green fluorescent at pH 8.0 (80). P a h t Phosphine (C.I. 789): for the region pH 6.0-7.0 ( 1 ) . Thiofauz'n (C.I. 816): for the region pH 6.5-7.0 (1). Umbellifemne: colorless at pH 6.5, blue fluorescent at pH 7.6 (30).
GH
As is evident, certain of the fluorescent indicators listed above exhibit two end points, e. g., quinine. I t is emphasized, however, that in some cases, especially the dyestuffs or little-studied indicators of a complex make-up, different brands or slight purity differences may explain the few discordant data of the literature, e. g., or-naphthionic acid, (3-naphthylamine, and erythrosin. In any event, there is much room for additional research in the field of fluorescent indicators. Literally, the fluorescence variation of an organic compound with pH is known in comparatively few instances when it is considered that several thousand fluorescent compounds aw known. LITERATURE CITED
( 1 ) BORGIALLI, A,, Bull. Uff. En. Stalz sperim. Ind. Pelli Materie Coneienli, 13,245 (1935). G. A.. Ind. Chem.. 4.8 (1929). 121 Acl-idine Orange (C.I. 788): orange colored at pH 8.4, green ~ -BRAVO. , , ( 3 ) BRENNECKE, ' E., "Neuere masan&tisehe Methoden," fluorescent at pH 10.4 (18, 13). Stuttgwt, Enke, 1952, Chap. 2. Ethor~lphaylnaphtho8tilbazmium chloride: green fluorescent , AND W. DIECK,Z. anal. Chem., 75, 81 (1928). ( 4 ) B u ~ o w C., a t pH 9, losing the emission at pH 11. ( 5 ) CANALS,E., Compt. rend., 198, 746 (1934). G Salt: dull blue fluorescence at pH 9.0, bright blue fluores( 6 ) CANALS,E., AND P. PEYROT,Seances Acad. Sci., 198, 1922 cence at pH 9.5 (12). (1935). Naphthazol derivatifies: stated to be colorless at pH 8.2, and ( 7 ) CLARK,W . M., "The Determination of Hydrogen Ions," yellow or green fluorescent at pH 10.0 (18). 2d. ed., Williams & Wilkins, Baltimore, 1923. a-Naphthionic aeid: blue fluorescent a t pH 9, green fluoresA. B., a m H. A. LUBES,J. Baete&d., 2, 1, 109, 191 ( 8 ) CLARK, cent at pH 11 ( l d ) . 119171. 8-Naphthol-3, Bdisulfonic acid: dark blue fluorescent at pH , ( 9 ) DE MENT, J., ''FI~orochernistr~,"Chemical Publishing 9.5, light blue fluorescent a t higher values ( I S ) . Co., New York, 1945. 1, 8-Naphlhobulfaic acid: dark blue fluoresoent a t pH 9.4, P., "Lumine8zenz-analyse im 6ltierten ultra(10) DANCKWORTT, changing to aligbter fluorescence at higher values (14). violleten Licht," Akad. Verlag., Leipzig, 1940. 8-Naohthol: colorless at DH 8.6. blue fluorescent a t h i ~ h e r 33, 364 (11) vines i14). . . DEL CAMPO, . A,,. AND F. SIERRA,Anal. Fis, Qnin., ~. (1935). a-Naphlholsuljonie acid: dark blue fluorescent a t pH 8.0, (12) DERIBERE,M., Ann. Chem. Anal., 18, 17 (1936); 18, 120 bright violet a t pH 9.0 (dQ). (1936); 18, 173 (1936); 19, 262 (1937); 19, 290 (1937); 8-Naphtholsuljmie acid: dark blue fluorescent at pH 9.0, Bull. assoe. chirn., 55,275 (1938). bright violet at pH 10.0 (89). 1, 4-Naphtholsuljmic acid: dark blue fluorescent a t pH 8.2, (13) DEGW,L. J., J. Am. Chem. Soc., 48, 1493 (1926). J., Pharm. Ztg., 75, 1033 (1930); Zeit. phys. (14) EISENBRAND, light blue at higher values (14). Chem., 144A, 441 (1935). Oreinsulfonohthaleinr vellow colored a t pH 8.6, fluorescent a t (15) FLECK,H. E., Analyst, 60.32 (1935). pH 10.0 (8). ' J., ibid., 56,653 (1931). Quinine (second end paint): violet fluorescent a t pH 9.5, color- (16) GRANT, (17) HAHN,F. L., Ind. Eng. Chem., Anal. Ed:, 14, 571 (1942). less at pH 10.0 (18,11, d6,14). %Salt: dull blue fluorescent at pH 9.0, bright blue fluorescent (18) HAITINGER,M., "Die Fluoi-eszenzandyse in der Mikrochemie," Haim, Leipeig, 1937. a t pH 9.5 (12). K. A,, Z. anal. Chem., 94, 177 (1933). Sodium 1-naphthol-2-suljonate: dark blue fluorescent at pH (19) JENSEN, ibid., 113, 326 (1938). (20) JONAS,J., AND L. SZEBELLEDY, 9.0, bright violet fluorescent at pH 10.0 (29). (21) Kocsrs, E. A,, AND K. BIRO,ibid., 122,94 (1941). pH 10 t o 12 (22) Kocsrs, E. A,, AND Z. NAGY,ibid., 110,317 (1937). Coumarin: deep green fluorescent a t pH 9.8, light green fluor- (23) Kocsrs, E. A,, AND P. VASS,Z. U n t m Lebensm., 71, 442 (1936). escence rtt pH 12. F., Ber., 9 , 1573 (1876). Eosin B N (C.I. 771): colorless at pH 10.5, taking on a yellow (24) KRUGER, T., J. SOC.Dyers & Colmrists, 52, 299 (1936). (25) MARSHALL, fluorescence at pH 14.0. R., A N D M. A. BISCHOFP,Compt. rend., 182, 1616 Papaveline (oxidieed by permanganate): yellow fluorescent a t (26) MELLOR, 11926) pH 9.5, blue fluorescent at pH 11.0 (do). \----,. Sehaeffers Salt: violet fluorescent at pH 5.0, green-blue fluore- (27) NASINI,A. G., et al., Atti del I 1 Cong. N w . Chim. pura appl., 7 , 668 (1929). scent s t pH 11.0 ( I d ) . Zauodsk. Lab.,3 , 1038 SS-Acid (sodium salt): violet fluorescent at pH 10.0, yellow (28) Punrmv, A,, AND M. S. MASLOVA, (1934). at pH 12.0 ( l d ) . J. A., AND J. GRANT,"Fluorescence Analysis in (29) RADLEY, pH 12 t o 14 Ultr*Violet Light," 3d. ed., D. Van Nostrand Co., New York. 1939. Cotamine: yellow fluorescent at pH 12.0, white at pH 13.0 120) -Ronr.. ~ . , Be?.. ~ 59. ~ 1725 , ~~11926). ~ , (18, 11, 86, 14). ~ - -z --R.. ~ N a p h t h i n i caeid: blue fluorescent at pH 12, green fluores (31) SALM,E., Z . phys. C h m . , 67, 471 (1901). Y., Awh. Phys. Biol., 6 , 61 (1928). (32) VOLMAR, cent at pH 13. Y., AND M. WIDDER,Bull. soe..chim., 43, 813 8-Naphthimic acid: blue fluorescent at pH 12, violetfluor- (33) VOLMAR,
pH 8 t o 10
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