(884) Vdovenko, V. M . , Krivokhatskii, A . S., Gusev, Y. K., Ibid., 2 , 531 (1960). (885) Vdovenko, V. XI., Krivokhatskii, A . S., Zh. .Yeorgan. Khini. 5 , 745 (1960). (886) Vdovenko, V. ll.]Lipovskii, A. A , , Sikitina, S. A . , Radiokhimiya 4 , 625 (1962). \ - - - - ,
(887) Vecera, Z., Bieber, B., Hut. n L i s t y 15, 667 (1961). (888) Veksler, R. I., Tr. p o Khim. z Khirn. Tekhnol. 1 , 142 (1962). 18891 Versteeen. J. LI. P. J.. Trans. Fara‘ day Soc. 58; 1878 (1962). ‘ (890) Verstegen, J. M. P. J., Ketelaar, J. A . A , , J . Phys. Chem. 6 6 , 21 (1962). (891) Vinarov, I. V., Orlova, A . I., Kishtsa, S. F., Ck. Khim. Z h . 28, 789 (1962). (892) Vinogradov, E E., Zh. Yeorgan. , 2813 (1962). Khzm. 77,”2813 (893) Vinokurova, G. N., l‘k. Khzm. Zh. 28, 651 (1962). (894) Voaliotti, Vogliotti, F., Energza AYucl.(Mzlan) 7 . 169 (1960). 0). (89;) von Baeckmann, A , , Glemser, O., 2. Anal. Chenc. 187, 429 (1962). (896) Vrchlabsky, SI., Okac, A , , Collection Czech. Cheni. Cornmun. 27, 246 (1962). (897) Vydra, F., Pribil, R., ChemistAnalyst 51, 76 (1962). (898) Wagner, W. F., Record Chem. Progr. 23, S o . 3, 155 (1962). (89!1) Waksmundski, A,, Socxewinski, E., Roczn. Cheni. 35, 1363 (1961). (900) Wang, Y. Y.,Khalkin, V. A., Radiokhirniya 3, 662 (1961). (901) Warren, C. G., J . Inorg. Sucl. Cheni. 23, 103 (1961). (902) Weaver, B., U. S. Atomic Energy Commission Report ORNL-3194 (1961). (903) Weglarczyk, A , , Chem. Anal. Warsaw 7 , 969 (1962). (9041 Weiss. D.. Rudu. Praaue 9 (4‘ Addendum) (1961). ” (905) Werner, .4. E., Waldichick, M., ANAL.CHEM.34, 1674 (1962). (906) West, T. S., Anal. Chim. Acta 25, 405 (1961). (907) West, T. S., Ind. Chemist 38, 35, 81 (1962). \ -
(908) Ibid., 38, 634 (1962). (909) West, P. W.,Lorica, A . S., ilnal. Chirn. ilcta 25, 28 (1961). 1910) Westoo. G.. Analust 88. 287 119631. (911) Wheali, R.‘D., B h d , B . J., Falania 9, 823 (1962). (912) Whitney, D C., U. S. Atomic Energy Comm. Rept. UCRL 1050 5 , 186 pp. (1962). (913) Whitney, D. C., Diamond, R. XI., J . Phys. Chern. 67, 209 (1963). 1914) Wilhelm. H. A , . U. S. Atomic Energy Comm. Rept.’ IS-309 (1961). (915) Wilson, A. I,., -4nalyst 87, 884 (1962). (916) Wilson, A . lI., Churchill, I,., Kiluk, K., Hovsepain, P., As.41,. CHEM. 34, 203 (1962). (917) Wilson, A. hI., hIcFarland, 0 . K., Ibid., 35, 302 11963). (918) Wilson, R. B., Jacobs, W. D., Ibid., 33, 1650 (1961). (919) Wish, I,., I b i d . , 34, 625 (1962). (920) LVoodward, I,. A , , Taylor, 3f. J., J . Cheni. Soc. 1962, 407. (921) Yagi, I., J . Chevk. Soc. Japan. Ind. Chews. Sect. 63. 1930 11060). (922) Ibtd., 64, 878 (i96l). (923) Yagodin, G. A,, Chekmarev, A . SI., Ekstraktsaua, Teoriua, Przmenenze, Aparatura 2,‘141 (1962). 1924) Yakimov. ;IT. A , , Sosova, N. F., Z i . S e o r g a n . Khim. 6, 208 (1961). (925) Yakovlev, I. I., Opalovskaya, R. I., Isv. Sibirsk Otd. -4kad. .I‘auli S S S R ( 1 2 ) 62 (1962). (926) Yamauchi, F., XIurata, A , , Japan Analyst 9 , !I59 (1960). (927) Yanagihara, T., Xfatano, S . , Kawase, A , , Ibid., 10, 414 (1961). 1928) Yeh. S.J.. Chu, P. C.. J . Chinese Chem. Soc. ( I I ) 10, 1 (1963). (929) Yen, T., Hsieh, Y . , Chem. Bull. Pekzng, 2, 17 (1960). (930) Yoshida, H., Japan Analyst 12, 169 i1963). (931) Yoshida, H., J . Inorg. .Vucl. Chenc. 24, 4257 (1062). 19321 Yosliida. H.. Takahashi. M., Jamn ~, Analyst 10, {I54 (1961). I-
/
,
‘
(933) Yoshida, H., Yamamoto, M . , Hikime, S.,Bunseki Kagaku 1 1 , 197 (1962). (934) Yoshimura, J., llurakami, Y., Bull. Chern. Soc. Japan 35, 1001 (1962). (935) Yuasa, T., Japan Analyst 10, 965 (1961). (936) Yuranova, 1,. I., Kommissarova, L. S . , Plyuschchev, T‘. E., Dokl. A k a d . .Yauk S S S R 140, 855 (1961). (937) Zaborenko, K. B., .%lian, .4., Zavod. Lab. 28, 1380 (1962). (938) Zagon, S. I.. Obogashch. Rud. 6, 3 2 (1961). (939) Zakharov-Sartsissov, 0.I., Ochkin, A . V., Zh. Seorgan. Khim. 7 , 665 (1962). (940) Zangen, J . Inorg: Sucl. Chem. 25, 581 (1963). 1941) Ibid.. D. 1051. (942j Zelle; A , , Fijalkowski, J., Chem. Anal., Warsaw 7, 317 (1962). (943) Zeman, A , , Ruzicka, J., Stary, J., Talanta 10, 685 (1963). (944) Zbid., p. 981. (945) Zharovskii, F. G., Sakhno, A. G., Ckr. Khim. Zh. 28. 145 11962). 1946) Zharovskii. F.’G.. Litvinenko. V. ‘ A,:Zh. Seorgan. Khim. 6 , 1940 (1661). (947) Ziegler, M., 2. Anal. Chem 182, 166 (1961) (948) Ziegler, M., Holland, J., Ibid., 194, 240 (1963). 1949) Zieder. 11..Matschke. H. D.. Ibid.. 184, 166 (196lf. (950) Zolotov, Y. A., Zavod. Lab. 28, 1404 (1962). (951) Zolotov, Y. A . , Alimarin, I. P., J . Inorg. jVucl. Chem., 25, 719 (1963). (952) Zolotov, Y. A., Alimarin, I. P., Radiokhimiya 4 , 272 (1962). (953) Ibid., Talanta 9, 891 (1962). (954) Zolotov, Y.A , Seryakova, I. V., Antipova-Karutaeva, I . I., Kut’senko, Y.I., Karyakin, A . V., Zh. ?;eorpan. Khiiii. 7 , I197 (1962). (955) Zolotov, Y.A , , Seryakova, I. V., Karyakin, -4.V.,Gribov, Id. -4., Zubrilina, 11.E . , Ibid., 8, 475, 481 (1963). (956) Zucal, R. H., Dean, J. A., Handley, T. H., A x . 4 ~ CHEM. . 35, 988 (1963). ~
Fluorometric Analysis Charles E. White, University o f Maryland, College Park, Md. Alfred Weissler,‘ Air Force O f f i c e o f Scientific Research, Washington 25, D. C.
T
review covers the 2-year period from approximately December 1961 (634)to December 1963. Several books, chapters, and a number of r e \’iews ’ on fluorescence have appeared in this period. Cdenfriend (499)in a 500-page book on “FluoreScence in I3iology and lledicine” gives d a t a on the fluorescence spectra of many compounds and discusses procedures and apparatus. Weissler and White (530) contribut,ed a chapter on fluorescence analysis in a “Handbook of ,\nalytical Chemistry’’ in which methods for 33 elements and 215 compounds are listed. The reagents, conditions, excitation and emission maxima, sensitivity, and references are given. HIS
1 Alfred Weissler is author of organic and biological section.
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ANALYTICAL CHEMISTRY
Nairn’s book (342) of 280 pages on protein tracing covers antibody techniques and material of interest for fluorescence applications in biology. T h e title of Gunn’s book (It?$), “Introduction to Fluorimetry,’’ is expressive of the content in its 59 pages. collection of the papers given a t the New York L-niversity International Conference on the Luminescence of Organic and Inorganic Materials is not concerned directly with analyses but does give considerable theoretical material from a physics and physical chemistry standpoint (236). Review 1)alm-s have appeared in many foreign journals. Parker and Rees (877) have given an excellent review covering theory, methods, and applications and include 150 references.
Parker (370) has also published a short review on phosphorimetry. Fluorometric analysis has been reviewed by Shcherbov (431j with 144 references; Patrovsky (378), 4 i references; Konstantinova-Shlezinger (262), 154 references; Holzbecher (213)) 120 references; Eisenbrand (141), 30 references; and Bozhevol’nov ( 6 6 ) , 122 references. Dorr (133) has written an excellent general article on the theory, equipment, and spectra determinations in fluorometric analysis. Crystalline luminous substances in inorganic analysis are the subject of an 8-page article with 68 references (224). 13owman (64)has a 3-page article on application of fluorescence to submicrogram analysis. An excellent 25-page review on the effects of environment, pH, concentration,
ionization, and solvent!, is given by Van Duuren (508). The relation b e t w e n fluorescence and absorption spectra of organic molecules has been t h e subject of a number of articles. I!4 7 1 ) . ,Ibsorption and fluorescence of pyrene and benzopyrene in variolis solvents are compared (5.57). hbso.*ption and emission spectra are compared and a modified equation relating these .spectra is derived (246). h review of the relationship of absorption to emission with 25 references is given by Kawski and Korba (241). Weber has presented a paper on the theory of fluorescence polarizations and its application to biochemical studies (526). Approximation formulae for concentration-dependent luminescence art discussed by I3uhrow ( 8 6 ) . A new technique of considerable promise in the measurement of fluorescence spectra has been reported by Schachter and Haenni ( 4 2 9 , who have devised instrumentation for the simultaneous recording of excitation and emission sliectra. Spe:tra of over 30 polynuclear hydrocarbms have been recorded by this technique and relations h i p of s1)ectra to structure have been found which were not rwealed by usual methods. Several significant reports have appeared on the correction of apparent fluorescence excitation and emission spectra. Parker (367) has shown that fluorescein may be used to calibrate spectrofluorometers for #?missionspectra in the ultraviolet region. .irgauer and White (15) have show:? that the aluminum chelate of 2,2‘-dihydroxy-l,1azona~1hthalene-4-sulfonicacid may be used as a fluorescence 5tandard for excitation spectra from 2!50 to 500 mp. This chelate is also a good addition to previously suggested €:mission standards, especially for regions from 500 to 700 mp. These authors have shown that emission spectra measured on the Aminco-13owman spwtrofluorometer withe a n RC.1 7102 electron multiplier phototube need little or no correction in the red region of the s.)ectrum. hlelhuish uses Rhodamine 1%as a quantum counter (315) and quinine sulfate and anthracene as standards (326). Drushel (134) and his associates have programmed the correction :’actors for computers and easily obtain the true excitation and emission curve:; as well as the
quantum efficiency. Proposals have been made for the qtandardization of methods of reporting fluorescence spectra (94]320). Parker (S69) has shown that sensitivity may be calculated in quanta emitted per quanta absorbed from the true excitation spectrum. H e gives d a t a for quinine bisulfate, Rhodamine 13, fluorescein, eosin, anthracene, and thiamine hydrochloride. -1grneral discussion on fluorescence spectrum measurements with respect to instrumental sensitivity i p given by Bowen (63). T h e effect of scattered light, including Rayleigh and Tynclall scattering, is important in fluorescence spectra measurements (992). Some of this may be corrected by filters (256). h geometrical aspect of fluorescence spectroscoliy is discussed by Thomas and his associates (486). The effect of substitution on the fluorescence of phenol and aniline has been reported for 1” values u11 t,o 14 by Rosen and Williams (406). Experiments on the effect, of halogen salts on the fluorescence of quinoline and various quinoline derivatives in acid solution show that the extinction of fluore-9cence by 13r- and I - decreases in the order of quinine sulfate, 6-methoxyquinoline, 8methoxyquinoline, and quinic acid. The order with C1- is the same, except that 8-methoxyquinoline showed the least extinction (142). Fluorometric grade solvents such as acids, alcohols, ether, ethyl acetate, N,lV-dirnethylformaniide, benzene, and toluene are commercially available (192). For phosphorimetry a n extremely useful tabulation has been made of the characteristics of nearly 90 solvents or solvent mixtures (539). Fluorescence on paper chromatograms is much intensified and phosphorescence is observed if the papers are immersed for several seconds in liquid nitrogen or liquid air and then examined under ultraviolet radiation (5.9). The sensitivity of detection of substances on paper chromatograms in shortwave ultraviolet radiation can be increased by spraying with a 0 . 0 0 5 ~ o solution of fluorescein in 90% ammoniacal ethanol or by a fluorescent screen prepared by suspending magnesium tungstate or fluorescein in 2% poly(viny1 alcohol) (479). APPARATUS
Commercial instruments for the measurement of fluorescence have improved and several additional models have been produced. The Aminco-13owman (10) spectrophotofluorometer is now designed for phosphorescence measurements at low temperatures as well as fluorescence at room temperatures. .in attachment to this instrument is available for the assay of solids, or for solutions from the
front surface of the cuvette. The improved xenon lamp is standard equipment. The Farrand spectrofluorometer (154) also has been improved with a xenon arc and better focusing with an ellipsoidal lens. The Perkin-Elmer Corp. has added a spectral fluorescence attachment to a double-beam spectrometer (381). This has been described by Porro (389). The Carl Zeiss (555) spectrofluorometer is formed by a n attachment to its PMQ spectrol)hotometer and is no\\- available from a representative in this country. The Beckman Instrument Co. has a n attachment for the DC spectrophotometer for t h e measurement of fluorescence ernission spectra (85). M a n y of the above firms making spectrofluorometers have also improved or presented new filter fluorometers. The .imerican Instrument Co. has designed a filter fluorometer which holds 20 samples in a rotary turret for correct positioning, and may also be used for Iihosphorescence measurements a t low temperatures (9). Ueckman Instruments has a new filter fluorometer (36) which uses the basic Beckman Dl3 circuit for measurement of the sample beam against a reference beam. This instrument has a n eight-sample turntable and is equipped for solids as well as for a flow-through sample. Technicon Instruments Corp. (484) and Turner Associates (496) fluorometers are also designed for continuous automatic operation. Hilger and Watt, Ltd., of England has a new four-sample filter fluorometer (206). Jouan Co. of France (232) and Kipp E n Zonen of Holland (250) both have filter fluorometers and are rep-esented in this country. Many research workers have constructed their own fluorometers and modified sliectrol~hotometers or other commercial instruments. Melhuish and Nurashige (317) have provided a double-beam attachment for the ;\mince-Kiers sliectrophosphorimeter so that excitation spectra can be recorded directly with a n accuracy of 10%. Parker and Hatchard (373) have introduced a pair of chop1)er disks into a s1)ectrofluorometer in order to eliminate fluorescence in the measurement of phosphorescence. Loeber has develolled a low tem1)erature absor1)tion cuvette for a spectrophotcmeter and a fluorescence spectrometer for the measurement of absorption and fluorescence spectra (285). Sill (445) has devised a simple, inexpensive accessory for the Cary JIodel 14 recording s~)ectrolihotometer, for the determination of excitation spectra. Hengge (201) has described a “universal” fluorescenee spectro1)hotorneter for the study of optically oliaque samliles. Shcherhov and I’onomarenko (440) have added a unit to a standard sl)ec,trolihotometer for recording both excitation and emission spectra. They VOL. 36, NO. 5, APRIL 1964
117 R
have also designed a simplified fluorometer (439) for routine work. The use of a discharge lamp in place of the mercury vapor lamp is advocated in order to increase sensitivity and reproducibility of a fluorometer (435). A microscope-spectrofluorometer (3) may be used for either solids or liquids. Arrhenius (18) calls his instrument a n ellipsoid fluorometer from its ellipsoidal cylindrical mirror, which has a reflecting roof and floor. Fletcher has described a vertical-axis transmission filter fluorometer for solutions (163),and has presented a n excellent discussion of fluorescence measurements in connection with this instrument (162). Simple photoelectric fluorometers have been designed by Bruskin ( 8 5 ) , Koziol (269), and Bakhirev and his associates (27). Apparatus has been constructed for thermoluminescence measurements (58) and a four-specimen liquid helium cryostat is designed for solids a t very low temperatures (284). Several new fluorometers have been designed to measure fluorescence decay of to second (Tb'), lO-'to second, (48), and by a stroboscopic method ( 8 2 ) . Fedorov and Freibart have described a double-beam fluorometer for measuring the luminescence of uranium beads (156). Takemoto (482) gives a block diagram for the modification of a spectrofluorometer for scanning paper strips. A polarization fluorometer and its use were the subject of a paper by Bromberg (80). A simple device for locating ultraviolet-absorbing substances by use of a fluorescent screen is described by Katz (240). A daylight detector is in use for finding fluorescenttraced spray3 on grain (464). Recording automatic instruments are available for titrations with fluorescence indicators (218, 476). Laikin has described a method of depositing a Sichrome mirror on a test tube for use in fluorescence measurements (276). The KBr pellet technique which is used in infrared analysis has been applied to fluorescence by Van Duuren and Bardi (509). This method is used to measure the spectra of solid aromatic compounds. Lithium hydroxide pellets have been used in a similar manner in a study of steroids ( I ) . I n this case R solution of the steroid was dropped onto the lithium hydroxide pellet. After evaporation of the solvent the spectrum was measured. Results are given for nine steroids. INORGANIC
General. A spray reagent consisting of 1 p a r t of 1Oy0 silver nitrate a n d 5 parts of 0.2% sodium fluoresceinate in absolute ethanol has been suggested for the detection of common organic a n d inorganic anions, es1 18 R *
ANALYTICAL CHEMISTRY
pecially those t h a t form insoluble silver salts (386). Absorption and fluorescence properties of 8-quinolinol compounds with elements in the second, third, and fourth groups of the periodic system have been determined (184). The authors present fluorescence spectra in chloroform, butylamine, water, and alcohol for 13 of these elements; Zn and dl give the greatest intensity and Hf and Cd the least. Ohnesorge and his associates (360) have continued their study of the fluorescence of 8quinolinol metal complexes and have shown that .Uf3forms a 1 to 1 chelate with 8-quinolinol in ethanol. The absorption and fluorescence spectra of 8-quinolinol and o-salicylideneaminophenol with the cations of Al, Ga, In, Zn, and Cu have been determined after extraction with chloroform and a mixture of chloroform and isoamyl alcohol, respectively (69). The same authors have shown that fluorescence intensities of metal complexes may be increased up to 1000-fold by cooling with liquid nitrogen (70). Bishol) (5.4) has reported on the p H requirements, complex formation, and use ae a titration indicator (53) of 8-quinolinol sulfonic acid with the metals usually determined with 8quinolinol. H e has also investigated the salicylate complexes of Cu, Ce, Xi, Zn, Mg, Be, Fe, and A1 for fluorescence properties ( 5 2 ) . Holzbecher (212) has determined the apparent aFsociation constants of All Sc, Zn, Be, and Ni ions complexed with salicylaldehyde, resorcylaldehyde, and gentisaldehyde. Absorption spectra, fluorescence spectra, and composition were determined. Badrinas (26) has made six new compounds by uniting o,o'-dihydroxyazo dyes and 8-quinolinol and has determined the color and fluorescence of the chelates with several cations. 1 1 1 of these compounds give a pink fluorescence with A l + 3 and Mg+2; some are very specific and extremely sensitive. This seems to be a welcome addition to our usual fluorescence reagents. Luminescence in the system SbzOr Sb20s can be used to detect small amounts (0.1 p.p.m.) of hIn. The ions of Ag, Sn, Ca, TI, 3x0,U, Ti, and Ce do not activate the luminescence. SbzOd is necessary for the luminescence. Boron and arsenic increase the intensity. Pb, Cd, Fe, Ni, Ce, and Cu have a quenching effect. Ba and Be prodirce new luminescent substances (8). The fluorescent materials zinc sulfide and fluorescein have been used as tracers on solids released in the atmosphere in fallout studies (290, 291). Przibram (394) postulates that the blue fluorescence, which is excited a t 365 m p and is widely distributed in water and minerals, is caused by traces of organic compounds absorbed on silica. This fluorescence is destroyed by heating
to redness and by acid treatment but is produced again by treatment with glycine. The fluorescence of sea water, measured at 546 mp, doubles between the surface and a depth of 100 to 200 meters and then remains almost constant to 2000 or 3000 meters (226). The fluorescence analysis of minerals has been reviewed by Shcherbov, who cited 142 references (430). Zeschke (556) gives a list of minerals with their fluorescence colors and describes field tests for C , Nb, W, and Be. Aluminum. Holzbecher (215) has shown that salicylaldehyde formylhydrazone serves as a fluorescence reagent for aluminum and that 0.7 to 22 pg. of A1 in 25 ml. may be determined with a n error of 1%; the limit is 0.08 pg. in 25 ml. Elements which do not interfere are listed and it is shown t h a t the interference of Fe, Ni, Zn, and Cr can be suppressed with thioglycolic acid. Small amounts of copper are masked with sodium thiosulfate. Paul and Gibson (380) have compared morin to the colorimetric reagents for the qualitative detection of aluminum and conclude that the morin test is the most satisfactory. Morin provides the end point indicator in the titration of aluminum with oxalate, with a relative error of about 3% for 100 pg. of aluminum in the analysis of minerals (476). Very, small amounts of aluminum in aoids and S i 0 2 are determined with 8quinolinol extraction in CHCls a t a limit of 0.02 p.p.m. (398). The rapid detection and approximate determination of aluminum in natural waters by the 8-quinolinol method are shown to be superior to colorimetric procedures (341). An interesting method for the determination of aluminum in industrial wastes uses sodium fluoride to prevent the extraction of aluminum with 8quinolinol. The aluminum ion is then liberated and the fluorescence of its complex measured (354). The 8-quinolinol method is used to determine traces of aluminum in uranium ( I & ? ) , bismuth (169), and lead salts (255). Ohnesorge and Burlingame (369) have shown t h a t the aluminum complex with 2-methyl-8quinolinol in absolute ethanol is more intensely fluorescent than the 8-quinolinol complex but is decomposed by 5% water. Danneil (113) prefers 8-quinolinol for the determination of micro amounts of .I1 in tungsten and tungstic oxide but uses Pontachrome B.B.R. for traces of A1 in zinc sulfide. Beryllium. Several authors h a v e reported improvements on t h e morin method for beryllium. These include t h e use of complexing agents t o exclude catalysts for t h e oxidation of complexing agents to mask morin (448), interfering ions (327), proper buffers (379), separation by the titanium phosphate method (481). and the use of Sn+* to prevent oxidation (329). Inter-
ferences are noted by the above and also by other authors (438). Efimychev (137) has shown that salicylaldehyde can be used as a n effective reagent for beryllium in acetone or ;amyl alcohol but not in water. Several of the azomethines have been tested as reagents for Be and (264). Lieryllium can be determined in bronze, without the removal of .U, Fe, or Cu, with 3-hydroxy2-naphthoic acid, provilled these metals are combined with a complexing agent (98). Boron. T h e benzoin method has been found useful for 1 to 30 pg. of boron in iron-containing raw materials; elements which either cause or quench the luminescence are also noted (436). Calcium, Copper, and Cobalt. T h e fluorometric determination of trace amounts of calcium in blood serum (244, 522) and urine (210) with the calceintype indicator has been successfully applied. The amounts of metals which interfere with this procedure have been determined (68) and potassium cyanide has been used to reduce the interference of Cu, Fe, and Zn (228) Calcein is also used in a n u1tramicrotii;ration of serum calcium (263). -4new reagent has been suggested for calcium, which is the bioluminescent protein aequorin. Light emission takes place upon the addition of C a ion and to a lesser extent with Sr ion. T h e aequorin is extracted from jellyfish. The sensitivity range is 0.01 pg. of C a + 2 in 2 ml. of solution (442). T h e fluorescence spectra of the alkaline earth derivatives of a number of azo compounds have been recorded by Olsen (363). Chemiluminescence methods with luminol and hydrogen peroxide have been developed for bclth copper and cobalt. Sensitivities are in the order of 3 X lo-* and 2 X gram, respectively (24, 25). Salicyl~ilazineand 2-(0hydroxyphenyl) benzoxitzole have been suggested as reagents for copper. The sensitivity of the former is 0.25 pg. in 5 ml. a t pH 12. Copper ions weaken the fluorescence of the second compound. Formulas for both com1:lexes have been determined (65). Gallium, Indium, and Gold. Rhodamine B finds extensi1.e use in t h e determination of gallium. It is shown t o be 10 times as sensitive as 8quinolinol a n d directions are given for its use in t h e analysis of minerals (613). It is used also in the analysis of by-products from the zinc industry (16). Mixtures of ?U's.cl and H 2 S 0 4 have no advantage over 1 to 1 HCl in this procedure (452). fhlgarian coals were found to vary from 0.0001 to 0.002170 Ga by the Rhodamine B (11). Exiwriments with method Rhodamine S as a reagent for gallium in metals have shown that even so-called pure salts of -41, Zn, Cu, and P b may contain to Ga. These re-
agents can be freed of gallium by extraction from 1 to 1 HCI solution with 9 parts of benzene and 1 part of ethyl ether (433). Gallium may be titrated with E D T A if morin is used as a n indicator (178). Shkrobot (449) has made a n extensive study of gallium and indium with the hydroxyflavanols and has shown t h a t the order of stability of the complexes is: Ga-morin > Ga-quercetin > In-quercetin > In-morin > Ga-rutin. Mukai (337) uses 2-methyl8-quinolinol to determine Ga in A1 samples. Korenman and Sheyanova (265) have described 8-mercaptoquinoline as a reagent for Zn, Ga, or I n . The complex is extracted with benzene. Sulfonaphtholresorcinol is recommended for the determination of G a in Zn and in Si (347). The complex of indium with 2 - methyl - 8 - quinolinol in absolute ethanol has been studied in some detail by Ohnesorge (368). Evidence for the composition of the complex is given and a table is included for the calculation of p H in alcohol. Rhodamine S is also recommended as a reagent for indium (22). Kojic acid (5-hydroxy-2-hydroxymethyl-1,4-pyrone) in neutral or slightly acidic solution gives an intense green fluorescence with auric ions. A p H from 5.7 to 6.8 is satisfactory and the test is linear for up to 50 pg. of gold in 50 ml. (339). Iron. An extinction of fluorescence method for t h e determination of iron(111) is based on its interference with t h e Pontachrome B.B.R. (2,2'-di-
hydroxyazonaphthalene-5-sodium sulfonate) reaction with aluminum. T h e decrease in fluorescence of t h e A1 complex is proportional to t h e iron concentration from 0.02 to 0.2 pg. per ml. The sensitivity is 3 times greater than the spectrophotometric method with 4,7 - diphenyl - 1,lO - phenanthroline (55). Lanthanides. An extensive s t u d y of t h e absorption a n d fluorescence spectra of benzoyl acetonate a n d dibenxoylmethide chelates of t h e lanthanides has been conducted by Crosby and Whan. Results on over 50 chelates have been reported (110, 632). Fluorescence spectra of coordinated H o , T h (109), and D y (108) are given. The fluorescent prol)erties of the lanthanides in vinyl polymers (157) and in phosphate-borate fusions (541) are also of interest. These phosphateborate fusions can be used to determine Gd, E u , and Sm (280). Traces of gadolinium in fluorite and in metallic thorium and beryllium in the order of 0.01 to 1 p.1i.m. can be determined by fluorescence spectroscoliy (491). The luminescence of gadolinium salts a t room and liquid air temperatures has been recorded (554). The fluorescence reactions of scandium have been studied with a number of o,o'-hydroxyazo
compounds and salicylidene-o-aminophenols (266). The effect of 44 common elements on the fluorometric determination of scandium with salicylaldehyde semicarbazide has been recorded (26.9). Scandium can be determined with 8quinolinol in the presence of other lanthanides (437). The absorption and fluorescence spectra of SmC13 a t 4' K. have been studied by Dieke and his associates (302). These authors have shown t h a t the fluorescence spectrum of S m + 2 resembles that of Eu+3 (122). An analytical procedure for cerium is based on the fluorescence of t h e Ce+3 ion and is said t o be accurate (=I= 10yo) for the range of 3 t o 100 X 10-6 gram ion per liter in the presence of Cef4 (17). The absorption, excitation, and emission spectra of E r f 3 in LaF3 have been recorded (273). Fleck (161) has made a spectrofluorometric study of t h e morin and 5,7-dichloro-8-quinolinolchelates of lanthanum. T h e fluorescence decay times of the benzoylacetonate and dibenzoylmethide chelates of Sm+3, E u + ~ , and T b + 3 are much longer than those of the salts of these ions (43). The fluorescence lifetimes of the europiumdibenzoylmethide chelates have also been studied by other authors (323, 415). -4trace of terbium is found t o increase the fluorescence intensiby of solid europium tungstate (510 ) ; the degree of enhancement is proportional to the T b concentration. The preparation and fluorescence spectra of europium tungstate are described by 1LlcI)onald and his associates (299). The absorption and fluorescence spectra of europium th,enoyltrifluoroacetonateand t h e fluorescent lifetimes of this chelate have been determined (180, 540). The absorption and fluorescence spectra of erbium chloride in lanthanum chloride have been determined by Dieke and Singh (123). Magnesium. After a n extensive s t u d y of t h e o,o'-dihydroxyazo compounds (964), Diehl and his associates have selected o,o'-dihydroxyazobenzene as a fluorometric reagent for magnesium; calcium shows but little interference (121). Babenko has shown that Tropeolin 000 is a sensitive reagent for magnesium (20). Schachter (422) uses 8-quinolinol-5-sulfonate in a fluorometric method for M g in place of 8quinolinol. Hill (207) employs 8-quinolinol in ethanol in a n automated method for the determination of Afg in serum. Niobium, Tantalum, and Rhenium. Lumogallion, 5-chloro-3-[2,4-dihydroxyphenylazo] - 2 - hydrosyhenzenesulfonic acid, forms a rose-red comlilex with ?;b(V) in 0 . 0 2 5 osalic acid ( 2 5 4 ) . The maximum intensity of the fluorescence is a t 625 to 630 m p with a 1 to 1 molar ratio. The intensity increases as the p H is increaFed to 5 and remains constant t o 6.5. The relation is linear for 0.1 t o 2.5 pg. of S b per ml. TanVOL. 36, NO. 5 , APRIL 1964
* 1 19 R
used as a fluorescence reagent for the tungstate ion. This will determine 0.5 to 4 p.p.m. with a n error of less than 5%; 5 ml. of 1% reagent is used in 50 ml. of water solution a t a p H of 4.8 to 6.2 (96). Uranium. T h e fluorescent method for t h e determination of uranium has been reviewed by Markov with 13 references (305). A new fluorometer for uranium determination with a beam splitter on the excitation is described by Haran (189). New equipment for making fluorescent melts has been designed by Lewandowski (282). An improved furnace for very small amounts of uranium, with six platinum dishes placed horizontally, is described (414). Applications of the fluorescent fluoridecarbonate melt to special cases of U determinations are given as follows: in air dusts (446), in minerals (169, 357, 480), in soil silts, plants, and animal tissue (348), in urine (459, 542), and in natural waters (14, 461). An improved extraction is described (362). The fluorometric method is about as accurate as the polarographic, but is more sensitive and more rapid (205). A visual comparison of the melt is claimed to be very rapid and reasonably accurate (450). A rapid fusion method with 25-mg. beads requires close temperature control for reproducible results (46). The effect of impurities in the fusion method shows that this can also be used to determine Ce and Xb (502). h paper chromatographic method for uranium involves a treatment on the paper with sodium phosphate, zinc acetate, and sodium acetate ( 5 , 176). Fluorescent solution methods for uraThallium, Thorium, Tritium, and nium are improved by the use of fluoride Tungsten. T h e nature of t h e lumiand phosphate along with the sulfate nescence of thallium(1) chloride has (129). The use of trimetaphosphate been elucidated by a study of its (130) also seems to have some adfluorescence a t various temperatures. vantage. Complexing agents for other T h e blue fluorescence originates in t h e metal ions and the absence of crystal lattice and is not due to acoxidizing and reducing agents are imtivation by water: the orange lumiportant in the solution method (117). nescence is caused by crystal defects The fluorescence spectra of nine difand not by colloids (457). Siloxene is ferent uranyl salts have been reinrecommended by Buzas and Erdy (90) vestigated a t room and liquid air temas a n end point indicator in t h e titration peratures (346). of thallium(1) chloride with dichromate. Zinc and Zirconium. Zinc may be Sill and Willis (447) have continued determined by t h e fluorescent comtheir studies with morin in the deterplex with Rhodamine S through a n mination of minute quantities of metals extraction from a thiocyanate solution and have given the conditions for the with ether. The reaction has a sensidetermination of submicrogram quantivity of 0.2 pg. per. ml. h p H of 3.5 tities of thorium. The proper choice of to 5 and the concentration of the thiobuffer and complesing agent is imcyanate are important ( 2 1 ) . .Inother portant in this method where a p H of 11 method following a thiocyanate exis used. The determination of tritium traction with isoamyl alcohol, and backn i t h a liquid scintillator solution of extraction into ammoniacal ammonium 0.5y0 diphenyloxazole and 100 mg. per liter of 1,4di-2-(5-phenyloxazolyl- chloride solution, uses a n ethanol solu8-(toluene-p-sulfonamido)tion of benzene) may be classed as a fluoresquinoline as the reagent (434). Equicence method (40). Tungsten in the molar salicylaldehyde and cyclohexylform of the tungstate ion gives a blue amine as a reagent in acetone will fluorescence with flavonol which is linear permit the extraction of zinc with amyl over the range of 6 to 42 pg. of tungsten alcohol and produce a good fluorescence in 100 ml. (60). Alizarin sulfonate is also
talum in a concentration 30 times that of N b has no effect; Ti or F e lowers the intensity; Zr, Hf, and citrates or tartrates do not interfere. Tantalum forms a fluorescent complex with the butyl ester of Rhodamine B and Rhodamine 6Zh. The complex is extracted from 10 to 12N sulfuric acid h i t h benzene. Ti, S b , and Zn do not interfere (66). Rhenium is also reported to give a fluorescence reaction with Rhodamine 6Zh, with a maximum emission from 530 to 535 mp in 0.5 to 1.5.V sulfuric acid. The fluorescent complex is extracted with benzene. Chromate, permanganate, and tungstate ions interfere. Determinations of R e in ores from 0.0002 to 0.1% are made with this method (224). Silicon and Selenium. Elliott and Radley have found t h a t benzoin, which has long been used as a reagent for boron, also berves as a reagent for silicon. The reaction is carried out in a n alkaline medium, with hydroxylamine hydrochloride present as a n antioxidant, and is sensitive to 2 pg. in 25 ml. of solution (143). The absorption and fluorescence spectra of siloxene and its methoxy derivatives have been recorded (200). Parker and Harvey (372) have proposed 2,3-diaminonaphthalene as a new fluorometric reagent for selenium. I t may be used in strongly acidic solution and is 20 times more sensitive than 3,3’-diaminobenzidine. This reagent has also been used by Lott and his associates (287). The application of 3,3’diaminobenzidine to the determination of selenium in plants has been studied with t a o new oxidizing agents (136).
120 R *
ANALYTICAL CHEMISTRY
(137). The association constants for Mg, Ca, and Zn ions with 8-quinolinol in 0.1M triethanolamine and tris(hydroxymethy1)aminomethane have been determined (624). Golovina and his associates have recommended quercetin as a reagent for zirconium on paper (174), and have shown t h a t datiscin (the 3-rutinoside of 3,5,7,2’-tetrahydroxyflavone)is a selective reagent for zirconium in 6N HC1 (173); the fluorescence is linear from 0.005 to 3 pg. of Zr per ml. Spectral studies on the fluorescence of 12 samples of zircons have shown t h a t dysprosium is responsible for three groups of identical lines in all of these (490). Chemiluminescence. A review article on chemical luminescence has been edited by Hayashi with 17 references (194). -1novel chemiluminescent system involves oxalyl chloride and hydrogen peroxide. A dioxane solution of oxalyl chloride added to a dioxane solution of 30% H202 containing anthracene produced a bright luminescence (93). Ojima (361) has demonstrated the chemiluminescence of several substituted monobenzoyl hydrazines in the presence of HzOa. The chemiluminescence of a,a’-azodiisobutyronitrile on oxidation in benzene is intensified by the addition of 9,lO-diphenylanthracene (514). Vasil’ev (512) gives intensity values for the effect of several fluorescent materials added to the chemiluminescent oxidation of cyclohexane in benzene. The addition of fluorescein to increase the intensity in luminol reactions has long been known but is now given a quantitative treatment (147). The acidity of dark oils may be determined with lucigenin (5,5’-biacridyl dimethonitrate) and H2O2in a 40 to 20 mixture of benzene and methanol on titration with 0.05N sodium methoxide (548). The chemiluminescence of lumino1 has been shown to be increased by cyanide (44) and inhibited by oximes (586). Low? level luminescence in nonaqueous titrations has been studied as an equivalence end point detection. An oxidation mechanism seems necessary to produce the luminescence even in acid-base titrations (127). The chemiluminescence of a number of organic compounds has been reported and offers some analytical possibility- for example, the oxidation of acetaldehyde (391) and the oxidation of urea, guanidine, and gelatin with hypochlorite (466). A system of measuring the chemiluminescent intensity of luminol in the determination of ferricyanide by the exposure to a photographic plate is sensitive to 0.3 pg. of ferricyanide in 20 ml. of solution (23).
Oxidation-Reduction and Acid-Base Indicators. I n addition to chemiluminescent redox indicators, two new fluorescent indicators have been de-
veloped. Rhodamine 6 G serves as a n indicator in t h e titration of uranium(1V) a n d vanadium(1V) with suIfatocerate(1V). At the equivalence point the greenish yellow of the fluorescence indicator is suddenly quenched with excess of the oxidant (124). Ruthenium( [1)2,2'-bipyridine complexes (272) give a n orange-red fluorescence, while thost. of Ru(II1) do not fluoresce; tris(2,:lf-bipyridine)-Ru (11) was found best for perchlorocerate titrations and tris(4,4'-dimethyl-2,2'bipyridine)-Ru(I1) was most satisfactory with sulfatot-erate and permanganate. A microluminescencs method for t h e titration of strong acids uses 2-naphthaquinone or p-umbelliferone with an instrumentally recorded end point (477). Plasmochin has been demonstrated to be a good indicator in the titration of strong acids and bases, and in iodometry (87). Metallofluorescent Indicators. E n d points in complexorietric titrations with fluorescent indicators a r e easier t o observe if t h e residual fluorescence is screened with a d:;e of a different fluorescence. I n t h e titration of calcium with E D T A and calcein or Calcein W as a n indicator, a few drops of a solution of O . O l ~ oacridine give a sharper end point. When Calcein Blue is used, Rhodamine B gives a n effective screen (252). A characterization study on the fluorescein complexon (fluorexone) indicator shows that the residual fluorescence a t p H 13 is caused by fluorescein, present as a n impurity. On this basis the fluoreicein content of the fluorexone indicator can be determined (67). Both 7-(2-hydroxy-4sulfonaphthylazo)-8-quinolinol with its analogs and bisglycint~-2,3-dichlorofluorescein are proposed as metallofluorochromic indicators for the chelometric titrations of Mg, Ca, C'u, Co, Xi, Fe, Cr, Zn, Cd, and V (309). The preparation and use of the two new metallofluorescent indicators 4,4 -diaminostilbeneK,N,N,'N'-tetraacetic acid and its 2,2'disulfonic acid have bl:en recommended for the titration of tha divalent ions of Cu, Co, Fe, Ni, and Pb as well as In(II1) and T.'(IV) ( 2 5 1 ) . The ions I-, I W , and (CX) may be titrated with
Rg+,
N,K-bis(carboxymethy1)amino-
methylfluorescein (fluorescein complexon) being used as tm indicator (520). ORGANIC AND I!IOLOGICAL
T h e continued grou th of fluorescence methods for organic and biological analyses has led to the publication of the books and reviews dizcussed a t the beginning of this paper. Aliphatic Compounds. Luminescence spectra have been used t o identify petroleum crudes (45, 338),
bitumens (ddb), lubricating oils (247, S l l ) , liquid paraffins ( I @ ) , and petrolatums and ceresin (139). I n hydrocarbon fuels, the free water content can be determined by filtration through paper impregnated with potassium fluoresceinate and measurement of the fluorescence acquired by the fuel (487). Virgin olive oil shows 60 little fluorescence that additions of refined olive oil (175) or olive-husk oil (118) can be detected by the increased emission under ultraviolet. Corn and soybean oils display a remarkable increase in fluorescence intensity (356) when heated to 170" C. Several aliphatic alcohols mere identified and semiquantitatively determined by the intensity of chemiluminescence obtained with 10, 10'-diphenylbiacridinium dinitrate (497). Low concentrations of formaldehyde can be analyzed by condensing with acetonylacetone and ammonium acetate, and measuring the fluorescence of the resulting 3,5-diacetyl1,Cdihydrolutidine (38); this has been applied to study the binding of formaldehyde to deoxyribonucleic acid. A highly sensitive method (420)for either formaldehyde or acrolein is to treat with J acid (6-amino-l-naphthol-3sulfonic acid) in sulfuric acid, and measure the resulting fluorescence a t 522 or 500 mp, respectively. Sawicki and associates have made comparative studies of procedures for determining traces of malonaldehyde, and conclude t h a t spectrophotofluorometry is most sensitive by far, using either p-nitroaniline (418) or 4,4'sulfonyldianiline (419) reagent. Several aldehydes can be identified chromatographically using their fluorescent azine derivatives (74). Ketone bodies in urine can be determined (after isolation by steam distillation) by their effect on the fluorescence of 2-naphthol (219). h microdetermination of alphaketo acids, based on the formation of fluorescent quinoxalines by reaction with o-phenylenediamine, is described by Spikner and T o a n e (460),who use a similar scheme for carbohydrates as well (489). X fluorometric method for blood glucose reported earlier has been adapted to pediatric work, where only small amounts of blood are available (62). Aromatic Hydrocarbons. Luminescence analysis for aromatic hydrocarbons has been discussed in detail by Parker and his associates (368, 370, 371, 374. 375, 376) and others (294, 472), with special reference to the delayed fluorescence effect. Flurrometric studies on 3,4-benzopyrene are being continued by a number of workers ( 4 1 , 125, 204, 349) because of its importance in carcinogenesis and air pollution. Very small traces of this compound, down to lo-" gram, can be determined with the help of the line structure luminescence a t 77" K (335,
336, 382). Ozone was found to quench the fluorescence of 3,4-benzopyrene (333); concentration quenching of pyrene derivatives has also been investigated (47, 132). Benzene, toluene, and p-xylene dissolved in several solvent's and a t various temperatures have been measured for fluorescence intensity as a function of concentration (227); similar studies on naphthalene (131) and anthracene (242) are also reported. The effect of excitation wavelength on the fluorescence efficiency of several alkylbenzenes has been studied in various states of aggregat'ion (239). Theoretical and experimental luminescence spectra have been reported for stilbene (281),2,6-dimethylnaphthalene (39), alkylanthracenes (516), fluorene and dibenzofuran (237), acetyl and amino derivatives of anthracene (97), several condensed ring compounds ( 1 4 5 & $ 4 ) and , mixed solutions of anthracene and p-terphenyl in benzene (478). Identification and determination by fluorescence spectroscopy have been proposed for a group of monomethyl derivat,ives of 1,2-benzanthracene (126) and for nine polycyclic aromatic hydrocarbons (138). I n many of the studies cited, it was found advantageous to work at, liquid nitrogen or even liquid helium temperatures. A detailed spectrophosphorimetric investigation of naphthalene, phenanthrene, and 1,2,4,5-tetramethylbenzene was made by McGlynn, Keely, and Seely (300). Aromatic scintillators such as terlihenyl have been studied (bo), with respect to fluorescence decay times (188) and quantum yield and oxygen quenching (199). Fluorescence spectra have been recorded for 13 subst'ituted polyenes (351). Polynuclear aza hydrocarbons are readily ident,ified by fluorescence spot test's after chromatographic separations (417). Ludwig and Herforth have studied the changes in fluorescence caused by the photolytic action of ultraviolet light, especially in solutions of aromatic and heterocyclic compounds (292, 293). Fluorescence properties of t'he tetracyanoethylene complexes of some alkylbenzenes have been compared (544). Other Aromatic Compounds. Spectrophotofluorometric studies have been done on benzaldehyde, other aromatic aldehydes and t,heir acetals (77, 112, 286,414). .inthraquinone can be determined by treating with glycerol, sulfuric acid, copper sulfat'e, and powdered zinc, and measuring the intensity of fluorescence of the benzanthrone formed (260). Luminescence spect'ra of the anthraquinonecarboxylic acids a t 77°K. shew changes when additional groups are introduced (429). aifluorometric determination of anthranilic acid maKes use of the decreased intensity obtained in the presence of glucose (416). VOL. 36, NO. 5, APRIL 1964
121 R
Mataga has studied solvent effects in t h e fluorescence spectra of the aminobenzoic acids and the naphthylamines (312). h group of papers presented a t the April 1963 meeting of the American Chemical Society dealt with solvent effects on the fluorescence of aminonaphthols (144), phosphorescence of o-phenanthroline and related heterocyclics (79), and fluorescence of monoand di-protonated o-phenanthroline (75) and of molecular and ionized 9-anthranol (366). Quinoline salts were found to show differing intensities of fluorescence in solution, depending on the nature of the anion (452). Solvent and pH efiects have been studied in the luminescence spectra of 5,6-benzoquinoline (248), and the fluorescence properties of mono- and polyazaindoles have been determined by Alder in order to provide a basis for development of analyt(ica1 methods ( 2 ) . Ultrasonic hydroxylation has been allplied by Weissler to the determination of benzoic acid (528) by measurement of the fluorescence intensity of the hydroxylated derivatives produced, principally gentisic acid. Similarly, ultrasonic hydroxylation of many substituted benzoic acids was found to yield fluorescent products of potential value for spectrophotofluorometric analysis (529). With the help of a new fluorescence method, homovanillic acid was identified in the caudate nucleus of the brain (4.28). Phosphorimetry as a means of chemical analysis has made a promising beginning (538) in providing a 10-minute analysis of aspirin in blood at concentrations down to 0.01 mg. per ml., using xenon arc excitation at liquid nitrogen temperature. Improved sensitivity in the fluorescence detection of o-hydroxy carbonyl compounds (such as methyl salicylate) was achieved by forming the boron chelates (441). The fluorescence spectra of several coumarins in various solutions have been determined (107, 233). The analysis of flavones (217) and other fluorescent compounds from spruce bark (268) has been described. .Steroids. Sensitive fluorometric procedures for analysis of steroids continue to be developed by many workers, based mainly on t h e fluorescence developed upon treatment with sulfuric or other acids. Analytical methods of this type have been discussed for aldosterone (196, 328), spironolactone, an anti-aldosterone steroid (395), free corticosteroids in plasma (76, 120, 313), estrone and other estrogens (31, 424), niethandrostenolone (488), and testosterone using glass paper chromatography (146). Hydrogen peroxide intensifies the fluorescence of est,rone in sulfuric acid, but, quenches that' of estradiol and estriol, thereby permitting a differential 122 R
ANALYTICAL CHEMISTRY
analysis (455). Cortisol and corticosterone can be distinguished by a preliminary extraction procedure which gets the former into aqueous solution and the latter int'o carbon tetrachloride (507). Additional fluorometric methods have been published for corticosteroids (170, 387) and for traces of progesterone
(444). For estrogens in urine or blood, greater specificity is achieved with the Kober-Ittrich procedure, which involves extraction after sulfuric acid treatment into a 2% solution of p-nitrophenol in methylene chloride or tetrachloroethane (275, 895, 410, 473). Total bile acids in serum can be determined, if cholesterol is removed, by fluorometry after reaction with sulfuric acid (279). Jakovljevic has described a new fluorometic method (using a reagent mixture of acet'ic anhydride, acetyl chloride, and trifluoroacetic acid) which gives a yellow fluorescence with digitoxin, but a lightblue fluorescence with digoxin (223). h peroxide-ascorbic acid spectrofluorometric analysis for these cardiotonic steroids is also available (531), as well as a n extensive review on the fluorescence reactions of digitalis compounds (165). Vitamins. Fluorometric methods for determining riboflavin, thiamine, 4-pyridoxic acid, a n d S-methylnicotinamide have been shortened without loss of accuracy by Starshov (465). For the microdetermination of riboflavine in blood, the fluorescence procedure based on photolyt,ic conversion to lumiflavine was found superior to other methods (546,551). Xicot'inamide analysis continues to make use of chloramine T oxidation and measurement of the resulting fluorescence (322). A good example of the value of spectrophotofluorometry is the diflerentiation among various coenzjme forms of folic acid: Folates do not interfere in the det,ermination of $10-methenyltetrahydrofolate at 360-mp excitation, 470-mp emission, if pH 3 is used (208); and at the same pH, by shifting the excitation to 300 mp and measuring fluorescence a t 360 mp, tetrahydrofolate itself can be determined without interference (505). Separate analyses for pyridoxine, pyridoxamine, Iiyridoxal, and their 5'-phosphates were achieved in the Iiresence of all six forms, by differential treatments with oxidase, transaminase, and phosphatase, before oxidation to 4-pyridoxic acid and lactonization to the fluorophore (343). I n t,he fluorometric Iirocedure for thiamine by alkaline ferricyanide oxidation to thiochrome, Haugen has pointed out several improvements in t'echnique (193). Errors due to high blanks can be minimized by reading the thiochroine fluorescence before and after dest'ruction by hydrochloric acid (251). Paper chromatography is a valuable adjunct
in the fluorometric determination of thiamine (37) and its phosphate esters (562). A modified thiochrome method suitable for cereals has been proposed (397), and the fluorescence and absorption spectra of riboflavin, thiamine, and thiochrome have been studied a t various p H values (226). Spectrophotofluorometry has made possible the simultaneous determination of thiamine and pyrithiamine; after ferricyanide oxidation, the fluorescence is measured a t both 435 and 480 mp and the results are calculated from two simultaneous equations ( 4 ) just as in spectrophotometric analysis of mixtures. Allternatively, thiamine can be separated from pyrithiamine by chromatography on polyethylene ~iowder before development of the fluorescence (401); neopyrithiamine can be determined in presence of thiamine by selective destruction of the latter with sodium hydroxide, before the ferricyanide oxidation step (402). A fluorometric procedure for 0-benzoylthiamine disulfide involves a preliminary reduction by cysteine, and use of cyanogen bromide to develop the fluorescence (503). Catechol Amines. I n t h e fluorometric determination of adrenaline and noradrenaline in biological materials, preliminary separations have been made using columns of alumina (12, 13, 203), Xmberlite IRC-50 (258), Dowex 50-X8 (453),and Sephadex G-25 (307). The ferricyanide oxidation of catecholamines was adapted to a semiautomatic fluorometric analysis for both noradrenaline and total catechol amines (321). Other oxidants such as L h O p have been used by various workers in the differential determination of adrenalin and noradrenalin by fluorescence (19, 234, 303, 403). Intermediate oxidation products which are precursorq of the trihydroxyindoles have been detected by spectrophotofluorometry (191). The condensation of catechol amines with formaldehyde to yield a fluorophore (152) has been utilized for histochemical demonstration of these substances in cerebral and adrenal tissues (57, 153). For the catechol amine metabolites, metanephrine and normetanephrine, Weil-Llalherbe and Sniith achieved differential analyses based on the principle of the trihydroxyindole method, by measuring the fluorescence after oxidation a t both pH 3 and pH 8 (4.54, 527). These metabolites iwhich are 3-methoxyadrenaline and 3-methoxynoradrenaline, respectively) may also be determined separately by using reaction rate differences: The fluorescence from the former rearhes its maximum within 10 minutes and then decreases rapidly, while the latter develops maximum fluorescence after one hour (396).
Other Amines. T h e yellow fluorescence of 5-hydroxytryptamine (serotonin) in 3!V HC1 continues to provide a useful quantittitive method for clinical determinations as in blood platelets, especially with improved preliminary separations (106, 111, 236). A better blank correction can be obtained hy taking the d.fference between the immediate fluorescence intensity and that remaining after 6 hours, by which time the serotonin will be destroyed (61). Increased sensitivity has been achieved with a ninhydrin condensation which gives an intense bluegreen fluorescence exceeding that of serotonin itself (506). A fluorometric method was useful cliiiically in following ~ ) l a s m alevels of demethylimipramine, a n active metabolite of the perot,onin antagonist imipramine (549). Tryptamine itsat,,is conveniently determined fluorometricdly ( 7 ) . A new procedure for 3-hydros,ytyramine (dopamine) is based on M n 0 2 oxidation and rearrangement t o a fluorescent compound with alkaline zinc sulfite (504); the fluorescence emitted is in the nearultraviolet around 385 mp, just as in the earlier dihydroxyinllole method (61). Improved separations make possible a fluorometric assay for histamine in urine (355). The histamine assay in blood, by o-phthalaldehyde condensation to a fluorophore, has won favor through its accuracy and simplicity (553). A specific fluorometric method for spermine and spermidine has been developed, based on t.heir osidation by amine oxidase to products which upon condensation with resorcinol give a fluorescence a t 520 InN (601). Ptomaines estracted from organs show intense fluorescence in HC1 solution (325). Uenzylhydrazine and other hydrazines in microgram amounts can be determined in blood plasma spectrofluorophotometrically, by their reduction of 1,2-naphthaquinone-4-wlfonic acid to the fluorophore 1,2-dibydroxynaphthalene-4-sulfonic acid (408). Drugs. Applicatims of fluorescence methods in pharmaceutical analysis have been discussed by several authors (83, 195, 493). A general method for detecting or determining amino or heterocyclic nitrogen is based on a reaction with a fluorescein to yield a more intensely fluorescent rhodamine dye (494):for example, atropine can be determined with high sensitivity by caondensation with eokin in chloroform solution and measuring the 580-mp fluoresrence produced (278). Another fluorometric method for atropine involves evaporation wi,:h HNO, to dryness, followed by reduction with zinc in S a O H solution (547). Belladonna leaves or tincture can be identified through the intense ‘due fluorescence shown by the chrysatropic acid resulting from the cleavage of the methylesculin
glycoside present in the plant (511). Both morphine and codeine in 0.liY H2SOaemit fluorescence a t 350 mp, but they can be determined simultaneously using the selective quenching of the morphine fluorescence a t p H 11 (28, 73). A highly specific and sensitive analysis for d-tubocurarine hydrochloride is to complex it with rose bengal in phosphate buffer, and measure the fluorescence after estraction into a (102). chloroform-phenol mixture Serum levels of pentobarbital were followed fluorometrically, after estraction first into chloroform, then into aqueous 0.2.U trisodium phosphate (211 ) . Ergotoxine and other natural alkaloids from ergot show intense fluorescence, which makes possible their determination as impurities in their hydrogenated (faintly fluorescent) derivatives such as dihydroergotoxine and dihydroergotamine (267, 536). A fluor6metric assay for yohimbine depends on its increase in fluorescence upon heating with hydrogen 1)eroside ( 9 9 ) . Emetine in animal tissues can be determined spectrophotofluorometrically by its intrinsic fluorescence a t 320 mp (116), or else by its golden luminescence after reaction with iodine in ethanol (221). ;it p H 1, antiamebic drugs derived from emetine show a fluorescence peak around 460 m p (618). For reserpine analyses, improvements have been made in the nitrow acid method (301); other fluorescence procedures have also been published (250, 267). A specific microfluorometric method for chlordiazepoxide (Librium) or its lactam metabolite involves hydrolysis to the N-oside, followed by exposure to bright light which causes rearrangement to a 4,j-eposide having a 480-mp fluorescence in alkaline solution (259). Mephenesin (71) and 4chloro-2-hydrosybenzbutylaniide (186) have enough intrinsic fluorescence so that their determination is straightforward. Fluorescence indicator paper has been used in the determination of bromides (466). To aid in the identification of quinine, its fluorescence characteristics in various solvents have been studied (283). A fluorescence analysis for isoniazid in serum has been described, based on oxidation with 30% H20Y(197). h spectrophotofluorometric method has been used for identifying 1)henothiazine drugs (318). Suitable for application to biological materials, fluorometric methods have been published for tetracycline (449), oxytetracycline and other derivatives, using a magnesium chelate (260). chlortetracycline using zinc chloride reagent (340) or degradation by alkali (412, 421 462), and pyrrolidinometh cycline (187). .\ procedure for mycin has been based on the intensity of fluorescence produced with 1,2-naphthoquinone-4-sulfonate in alkaline solu~
tion (155). Quantitative determinations of rotenone in derris preparations were performed by inducing fluorescence through ultraviolet irradiation of the aqueous or chloroform solution (550). In a n organophosphorus insecticide, the highly toxic thiol i:.omer was distinguished from the thion isomer by the thiol’s quenching action on the fluorescence of eosin (492). Amino Acids and Proteins. Phenylalanine levels in the serum of phenylketonuric patients have been followed by a fluorometric method (296, 545). Fluorinated phenylalanines were found to fluoresce much more intensely at 292 mp than phenylalanine itself or the chloro or bromo compounds (183). Detailed studies are continuing on the luminescence spectra of tryptophan and tyrosine, with attention to the effects of pH,liquid nitrogen temperature, and neighboring structures in the protein molecule (30, 104, 105, 519, 535). Histidine and tryptophan (and proteins containing them) developed a fluorescence in the visible, upon treatment with S-bromosuccinimide (72). The protein content of milk, after dilutionnith citrate-phosphate-urea,was determined accurately and rapidly by the fluorescence intensity in the ultraviolet (164): another fluorometric procedure for protein in milk is given by D’Yachenko (545). For trace amounts of protein, measurement of the quenching of Eosin Y fluorescence has been proposed (209). Lipoprotein fractions separated by electrophoresis can be determined by fluorescence intensity readings, after staining with protoporphyrin IX (425); similarly, total phospholipides from tissue have been determined fluorometrically, after complexing with Rhodamine 6G (190). A quantitative microfluorometric technique was described for anal fractions isolated from a mycobacterium (390). Steiner and Edelhoch have pointed out the value of changes in fluorescence spectra and polarization as a sensitive indicator of change in the structure of a dissolved protein (469), as in the case of soybean trypsin inhibitor (470). Prior conjugation of a fluorescent dye to the protein facilitated the detection of changes in fluorescence polarization, as applied to structure studies on solutions of ribonuclease (552) and lysozyme after reduction and air-reoxidation (100). Enzymes and Nucleotides. A convenient approach t o t h e assay of enzymes is measuring the amount of fluorescence libcrated from a suitable fluorogenic substrate. This has been applied in the determination of 1il)ase @ ? I ) , trypsin (407),fibrinolytic activity using fluorescein-labeled fibrin (365). alkaline phosphatase and aminopeptidase ( 1 7 7 ) ,and beta-u-galacto*iciaw and VOL. 36, NO. 5 , APRIL 1964
123 R
phosphatase (409). h related technique was used in getting evidence that flavine is the prosthetic group of succinic dehydrogenase (91), and in a fluoromet'ric determination of the content of this dehydrogenase in heart muscle mitochondria (451). Lowry and coworkers have devised a powerful method of broad applicability for assay of traces of enzymes down to possibly a single molecule, using a fluorometric measurement of pyridine nucleotides after a single or double stage of enzymatic cycling (288). Considerable interest continues in the fluorescence of the pyridine and other nucleotides (149, 150, 621), as further attested by two recent reviews on their fluorometric analysis (181, 345). h micromethod for the determination of acetylpyridine a d e n h e dinucleotide and its phosphate in brain is based on the fluorescence intensity after condensation with ethyl methyl ketone in alkali (202). The fluorescence of adenosine, its phosphates, and adenine was studied as a function of pH, and Mg+*and C a + 2 were found to bind to the phosphate moiety, not to the ring (523). Udenfriend has investigated the fluorescence of purines and pyrimidines in detail, and has given a fluorometric assay for dimethylaminoguanine in t'he presence of guanine, by destroying the latter with nitrous acid (498,500). Porphyrins. Luminescence properties of porphyrins have been discussed by various authors (128, 427, 458), and Hoschek has described a rapid, semiquantitative fluorometric determination of porphyrin in urine (216). Falk has reviewed the chromatography of porphyrins and metalloporphyrins (151). Hemin has been shown t o be a chemiluminescent substance (148). In blood, bile, or urine, determinations of urobilin were based on the fluorescence intensity after FeC13 osidation and addition of ethanolic zinc acetate (289, 411). Biliverdin can be converted into the zinc salt of bilipurpurin which has a red fluorescence (404). Fluorescent Staining. T h e specific identification of proteins or microorganisms by their immunochemical reaction with fluorescence-labeled antibodies has become so valuable that a book has been published on this and other aspects of fluorescent protein tracing (542). Efforts continue to be expended on such improvements as selecting the brightest fluorochromes for the labeling (103, 171, 172, 388), and removal of non.;pecific staining material by column chromatography (92. 298, 400). Fluorescein isocyanate was found to give twice the emission intensity of the isothiocyanate, in the case of the rabbit serum antibody against bovine serum albumin (298). The fluorescent antibody technique has been used in studying or identifying 124 R
ANALYTICAL CHEMISTRY
nonspecific thyroiditis (319) human submaxillary gland mucin (243),human and simian malaria (274), mouse mammary tumor agent (81), various malignant human tumors (95), renal lesions c lupus erythematosus (88), nee of a liver-specific antigen in hepatomas (160), hog cholera virus (463), equine encephalomyelitis virus (324), poliomyelitis antibodies by an indirect method (399), pleuropneumonia-like organisms in human tissues (101), single cells of Shigella .flerneric (115 ) , trachoma, psittacosis, and lymphogranuloma venereum (350), enteropathogenic E . coli in infant stools (308), various mycobacteria and corynebacteria (535). treponemal diagnostic tests (495), and localization of specific antigens in P a r a m e c i u m aurelia (33). Konantigenic fluorescent staining has found additional uses (119, 167, 326, 426) 515) particularly by the use of *kcridine Orange (29, 42, 135, 179, 306). I n a quantitative immunofluorescent titration of gamma-globulin, the excess fluorescent antibody was measured in the supernatant of the precipitation reaction niisture (485). The reaction between fluorescent antigens and their antibodies vias measured directly and sensitively by the extent of polarization of the fluorescence (185). The preparation of fluorescein sulfonic acids has been described (223). Miscellaneous. Russian workers have shown some interest in t h e intrinsic fluorescence of living cells (84, 26'1, 383). Characteristic primary fluorescences were a k o found in normal and pathologic keratin (468). The relation between chlorophyll fluorescence and photosynthesis has been studied in somedetail (89,413,483,517). I n tobacco plants, fluorescence has been used a:: a measure of quality (297) and for the detection of blue mold infection (34). Fluorescence analysis has been used in the medicolegal identification of blood (168, 249), soap on skin or cloth ( % I ) , seminal fluid or milk (332), and cosmetics (330 . I t has also been used in of essential oils and flavors (385),in the rubber industry (393), and t o detect rodent urine in grain (245). Fluorescence appearing in packaged chicken was found to be an indication of bacterial growth (270). h n interesting application of fluorescence is in the tracing of air currents (406) and of natural waters (304) using Rhodamine 13. The optical brighteners used for textiles and papers have been discused from several different aspects (114, 198,277, 310, 334, 637). Chemiluniinescencaehas been studied in various osidation reactions (467). The fluorescence of 14 polynier types such a:: polyethj.lene and poly(pheny1acetylene) has been described by Gachkovskif (166), who suggests its use in ~
molecular 1%eight determinations. Changes in the fluorescence spectra of a variety of cellulosic polymers were correlated n i t h modificationa in the chemical structure (384). The effect of styrene monomer on the fluorescence of polystyrene has been measured (32), and a fluorebcence polarization method has been used to study the internal structure of dye-labeled polymers in solution (235, 3 3 ) . LITERATURE CITED
(1) Abraham, R., Staudinger, H., Z . Saturforsch. 18b, 421 (1963). (2) Adler, T. K., ANAL. CHEM.34, 685 (1962). (3) Agroskin, L. S., Korolev, 1. V., Bzofizzka 6, 478 (1961). (4) Airth, R. L., Foerster, G. E., Anal. Bzochem. 3.383 11962). (5) Alberti, 'G., Saini,' A,, Anal. Chim. Acta 28, 536 (1963). (6) Alentsev, 11. S., Pakhomycheva, L. A., Opt. i Spektroskopiya 12, 565 (1962). (7) Allgen, L. G., Funke, K. E., Kauckhoff. B.. Scand. J . Clin. Lab. Invest. 13. 390 (1961) (8: .kllsalu, 11.L . Yu., V c h . Z a p . Tartusk. Gost. 17niv.1960, KO.95, 198. (9) American Instrument Co., Silver Spring, )Id., Nfr. Bull. 2390 (1963). (10) Ibid., 2392 (1963). (11) Angelova. V.. Koleva. D.. Ruschev. D., Godishnik K h i m . Tekhnol. Inst. 8; 1953 (1961). (12) Antognetti, R. RI., Kava, S., Boll. SOC.I t a / . Biol. Sper. 38 (23), 1176 (1962). (13) Anton, A. H., Sayre, D. F., J. Pharmacol. Exptl. Therap. 138, 360 11963). (14) Aoyama, Y., ;Vzppon Kagaku Zasshi 82, 336 (1961). (15) Argauer, R., White, C. E., 11th Inachem. Conf., Detroit, Mich., Oct. 23, 1963. (16) Armeanu, V., Costinescu, P., Bul. Inst. Poizteh. Iasz 8, 123 (1962). (17) Armstrong, W , A,, Grant, D. W., Hempryes, W. G., ASAL. CHEM.35, 1300 (1963). (18' Arrhenius, S., Arkiv Kemz 18, 165 (1961). (19) Atkinson, A , , Wynne, PI'. A., J . Pharnz. Pharmacol. 14, 794 (1962). (20) Babenko, 0. S.,Kauk Zap. Chernivets'k. Cnio. 33, 94 (1959). (21) Babko, A. K., Chalaya, Z. I., Z h . Analit. K h i m ; 17, 286 (1962). (22) Ibid., 18, a i 0 (1963). (23) Babko, A. K., Kalinichenko, I. E., t 7 k r , K h i m . Z h . 29, 527 (1963). (24) Babko, A. K., Lukovskaya, S . lf., J . Anal. Chem. Z'SSR 17, 47 (1962) (Eng. Trans.). (25) Babko, A. K., Tukovs'ka, S . hl., Zavodsk. Lab. 29, 404 (1963). (26) Badrinas, A,, Talanta 10, 704 (1963). (27) Bakhirev, S . F., SventitskiI, 1. I., Andreitsev., A. P., Butov, G. P., USSR Patent 144,047 (Jan. 27, 1962); C A 56, 4044 (1962). (28) Balatre, P., Claeys, C., Decamhre, J.-P., Traisnel, l l . , Bull. Soc. Chim. France (8-9) 1654 (1962). (28) Bancher, E., Hoelzl, J., Protoplasma 57, 33 (1963). (30) Barenboim, G. )I., Biojkika 7, 227 (1962). 131) Barlow, J. J., A n a l . Riochem. 6, 435 (1963). (32) Basile, Louis J., J . Chem. Phys. 36, 2204 ( 1962). ~
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(50) Birks, J . B., Geake, J. E., Lumb, A I , I)., Ijrit. J . -4ppl. Pliys. 14, 141 (1963). (51) I T r . Kazukhsk. A\-o uchn.-Issled. Inst. Mineral'n. Syr'ya j.961, S o . 5, 265; C'A 58, 11954 (1963) (57) Boner, A,, Langwnann, H., Saturwissenschaflen 50, 255 (1963). (58) 13onfiglioli, G., Brovetto, P., Cortese, C., Rei,. Scz. Instr. 3.3,1095 (1962). (59) 13orodin, S. S.,Galashin, E. A , Seniyakina, S. -4.:Silaeva, T'. S . , JI etotlij L y u miriestsii . A naliza, Sborn i k 1960,81 ; CA 56,4071 (1962). (60) Uotelli, It. S., Trusk, A , , Asai,. CHEM.35, 1910 (1962). (61) l
