Fluorometric analysis - ACS Publications - American Chemical Society

PSR Zinat. Akad. 2,47 (1965); CA. 66, S724p. (2673) Zamfirescu-Gheorghin,M., Vla- descu, C., Apostolesu, I., Chirulescu,. Z., Grosu, E., Microchem. J...
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(2670) %nl)olotnyiik,I. P., Ibid., 38, 357 (1966); Crl 66, 1014~. (2671) Znk, B., Wciner, L. M.> Baginski, IC., J . Chro?,rafog. 20, 157 (1065). (2672) Zakenfelds, G., Klin, Eksp. Aled., Lnlv. PSI? Zinat. Akad. 2, 47 (196.5); C A

66,8724~. (2673) Zmufirescu-Gheorghin, AI., Vladescii, C., Apostoleaii, I., Chirulescu, %., Grosu, E., iUicrochem. J . 10, 106 (1966). (2674) Zarkadas, C. G., Baker, B. E., J . Sei. Food Agr. 16,729 (1965). ( 2 6 7 3 ) Zarkadas, C. G., Henneberry, C;. I)., Baker, B. E., Ibzd., p. 734. (26iG) Zazvorka, Z., Kamaryt, J., Casopis I.rkaiu Ceskych 104, 970 (1963); C A 65, 5756f. (2677) Zec, J., illicrochenz. J . 9, 510 (1‘363). (2678) Zemlyakova, Z. Al., Telesheva, V. A,, dlateiialy /-ai [Pervoi] iYazcchn. K n o f . Tsenfr.Xaztchn.-Issled. Lab. Tomskogo Jled. Inst. Sb. 1964, p. 189; C A 63, 16930e. (2679) Zeya, 13. I., Spitznagel, J. K., J . Uacteriol.91, 750 (1966); CA 64,99819. (2680) Ibid., p. 755; C A 64,9981h. (2681) Zeya, 13. I., Spitznagel, J. K.,

Schwab, J. H., Proc. SOC.Exptl. Biol. fired. 121,250(1966). (2682) Zhidovtsev, V. M., V o p Eksp. Klin.

Radial., Khar’kovsk. Inst. Med. Radiol. 1, 105 (1965);C A 65,9517g. (2683) Zhiiravlev, G. I., Avgustinik, A. I., Vigdergaiiz, V. S., Kolloid. Zh. 28, 610 (1966);C A 66,5996k. (2684) Zicgenbein, R., Vogt, R., Arendt, A., Deut. Gesundheitsw. 21, 1685 (1966); C A 66,349301. (2683) Zimmermann-Gorska, I., Bull. SOC. -4nizs Scz. Left. Poznan, Ser. C 14, 71 (1965);C A 63, 16872e. (2686) Zingale, S. B., Manni, J. A., RIattioli, C. A., Acta Phipiol. Latinoam. 15,325 (1963);CA 64, ,54269.. (2687) Zinkham, W. H., Kiipchyk, L., Blanco, A,, Isensee, H., Sature 208,

284 (1963).

(2688)’Zittle, C. A., J . Dairy Sei. 48,

1149 (1965). (2689) Zittle, C. A., Custer, J. H., Ibid., 49,788 (1966). (2690) Zlotnick, A., Landau, S., J . Lab. Clin.Aled. 68,70 (1966). (2691) Zonta, A., Dionigi, R., Galli, E., Boll. Sac. Ital. Biol. Sper. 40, 1964 (1964); CA 64,5423d.

(2692) Zotov, V. A., Tr. Saratov. 2ootekh.Vet. Inst. 13,229 (1965); CA 66,8373.k (2693) Zueva, A. M., Dokl. T S K h A (Timiryazev, Sel’skokhoz. Akad.) 120, 61 (1966); C A 66,83753j. (2694) Zui, V. D., Visn. Kiiv. Univ., Ser., Ser. Biol. 8,130 (1966); CA 67,9044h. (269.5) Zwaan, J., Anal. Biochem. 15. 369 (1966). (2696) Zwaan, J., Exptl. Eye Res. 5 , 267 (1966). (2697) Zwartz, J. A., Bibliotheca Dieta 7,221 (1965);C A 64,72799. (2698) Zweig, G., Whitaker, J. R., “Paper Chromatography and Electrophoresis. Vol. 1: Electrophoresis in Stabilizing Rledia,” Academic, New York. 1967. 420 pp. (2699) Zwisler, O., 2. Physiol. Chem. 343, 178 (1965);C A 64, 14.503~. (2700) Zwider, O., Biel, H., Protides Biol. Fluids, Proc. Col/og. 12, 433 (1964) (Pub. 1965); C A 65,4243h. (2701) Zwisler, O., Biel, H., 2. Klilz. Chem. 4,38 1966);CA 65,2606d. (2702) Zydeck, F. A,, Muirhead, E. E., Schneider, H., Am. J . Clin. Pathol. 45, 323 (1966). (2703) Zyl, A. van, Wilson, B., S . African J . Lab. Clin. Med. 10, 15 (1964); C A 64,20319~.

Fluorometric Analysis Charles E. White, Universify of Maryland, College Park,

Md.

20742

Alfred Weissler,’ Food and Drug Administration, Washingfon, D. C.

T

is the eleventh of a series of biennial reviews on Fluorometric Analysis and covers the period from approximately Deccmber 1965 to December 1967 (706). During this period several books and a number of reviews have added greatly to the literature on the subject. The English transletioii of the 3i5 page book by Iionst’antinova-Shlezinger on “Fluorometric Analysis” is especially good on inorganic aiialysis (365). This book includes many tables of formulas which give the chemical as well as t,rivial names found in abstracts from Russian articles. A number of experiments are also outlined. Another book from Russia by E. A. Uozhevol’nor not yet translated, has a title which in English is equivalent to “Fluorometric Analysis of Inorganic Materials” (80). This book of 415 pages should be authoritative for the author is estremely productive in the area of research for inorganic ions. A review in English indicates that. this book is a valuable addition to the literature on fluorescence analysis (329). Professor J. Eisenbrand of Germany has published a 153page book on “Fluorimetry” which covers inorganic, organic, and biocheniical matcrials as well as geiieral theory and apparatus (194). A “Handbook of HIS REVIEW

1 Alfred Weissler is author of the Organic and Biological Section.

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20204

Fluorescelice Spectra of Aromatic Rlolecules” by I. 13. Berlman (51) gives absorption and corrected emission curves for 100 aromatic compounds. The symposia 011 Fluorescence Analysis a t the American Chemical Society Meetings of September 1964 and April 1967 have appeared in book form. The former is edited by D. AI. Hercules and contains chapters by eight authors (282). The latter is edited by G. G. Guilbault and has 23 contributors (260). Both of these compilations are good additions to a library of fluorescence analybis. “Guide to Fluorescence Literature” by R . A. Passwater (50281) assembles the references on fluorescence from 1950 to 1964 and includes 5000 titles which are cross referenced as to both authorq and subject. The title of Goldberg’s book “Luniiiiescence of Inorganic Solids” is descriptive of its contents and while useful is not designed for the aiialytical chemist (249). Likewise the very abbreviated books “Luminescence’ s’ols. 1 and 2 in Russian (540) are largely devoted to solid materials as phosphorq. A book on “Luminescence” in French by G. RIonod-Herzen 1450) of 278 pages and subtitled “The Electron and Luminophor and Photoluminescence” is not intended for the analyst. A chapter on “Chemical Spectrometry” by T. S. West, which contains

excellent material on fluorescence analysis is included in a United States National 13ureau of Standards monograph (702). General chapters on “Fluorometric Analysis” (700), “Fluorometry and Phosphorimetry” (699),and “Spectrometry and Fluorometry” (123) are included in standard reference books. Monthly bulletins of two instrument makers are devoted to current references and short articles on fluorescence analysis are free on request. The American Instrunlent Co. initiated its publication, Fluorescence Xews in February 1966 with an article on the effect of temperature on fluorescence emission and a number of references on fluorescence analysis ( I S ) . I n subsequent issues the editors discuss topics on fluorescence as, the effect of pH, the effect of solvent, attachments of the Aminco-Don man spectrofluorometer for corrected spectra (,503), and list reagents for 47 elements (500). -4similar bulletin called Traces coiisisting largely of current references has been published for several years by Turner Instrument Co. (663). One issue of this publication lists sources of reagents for the clinical procedures given in the “Turner Manual of Fluorometric Clinical Procedures” (664). -1 staff report in Cheniical and Engineering S e w s has given an historical review of the growth of Fluorometry in Analysis (114) and Hercules (28281) has reviewed present

generalizations on fluorescence and phosphorescence analysis. Reviews on fluorescence have been given in Germany by G. von Loeber with 270 references (404)and Fuss (227); in Japan by Shigematsu with 47 references (605) and Yagi and Nagata with 62 references (783); and in England by Parker (493). A survey of luminescence of complex molecules, based on the Soviet-satellite open sources, published in 1963, is available in a 113-page report of Kourilo (369). This report deals largely with solid state luminescence and only about six pages are devoted to analytical methods, most of which have been noted in a previous review (706). Schulman (580) has given an excellent eight-page review of luminescence in solids which deals chiefly with inorganic phosphors. The theory of luminescence and its use in various fields is the subject of a short paper by Bowen and Garlick (77). The fates of electronic excitation energy is the subject of an excellent article by JaffE and Miller (316). This article is well illustrated with diadorams and gives explanations of terms such as excinier fluorescence. A study of the relation of the emission spectrum to the excitation spectrum in solid solutions shows a complete independence in many cases (352) but a dependence in others (331,355). The substitution of deuterium for proton hydrogen in chromophor groups is shown by Stryer (637) to have a considerable effect on emission spectra and on quantum yield. The spectral dependency of absolute quantum yield in mixed solvents is discussed by Viktorova (679, 680) with 30 references. The importance of mixed solvents is demonstrated in this research where two compounds showed no fluorescence in either acetone or n-heuane but produced a strong fluorescence in a mixture of the two solvents. The use of quinine sulfate as a quantum yield standard has been studied by several authors. Drobnik and Yeargers (185) indicate that the widely used value of 0.55 as the quantum yield for quinine sulfate might be too high and should be near 0.4. Eastnian (190) shows values of 0.577 for an excitation a t 350 mp and 0.582 a t 250 m p for the quantum yield of quinine sulfate. Chen (116) shows that the quantum yield of quinine sulfate is 23% higher a t 390 mp than a t 313 mp excitation. He also states that the excitation spectra of quinine sulfate in acid solution fails to coincide with the absorption spectra a t long wavelengths and that the emission spectra of quinine and 6-methoxyquinoline are shifted more t o the red when excited a t 390 nip rather than near 340 mp. Fletcher (217) has Ptudied the quantum yield of quinine sulfate and 2-aminopyrine a t 298' K and 160' K. Studies on the luminescence and photochemistry of azomethine compounds (357, 374, 481) and of

quinolinols (841) should be of interest in both organic and inorganic analysis. I n a discussion of organic trace analysis, Parker (494) shows the importance of the purity of solvents. Kordan (366) draws attention to the fluorescent contaminants from rubber, bakelite, polyethylene, and other plastics. Crosby and Aharonson (145) show that many impurities may be removed by passage through charcoal and distillation. APPARATUS

Many of the instrument companies have improved their offering in the realm of fluorescence measurement. The iimerican Instrument Co. (18) has a new energy corrected attachment of the Aminco-Bowman spectrofluorometer which is claimed to fully compensate for light sources, monochromators, and photomultiplier tubes. A new light gathering system, a holder for pellets, and an attachment for reducing large chromatographic plates are some of the several items available for this instrument. The Turner Instrument Co. has also added scanners for plates and strips as attachments to their Models 110 and 111 instruments and have developed an absolute spectrofluorometer RIodel 210 (662) for recording absolute fluorescence spectra. The Farrand Optical Co. has a solid sample holder for pellets and a plate adapter for their ME;-1 fluorometer (208), and are reported to have attachments in progress for constant energy. An excellent description of the Farrand blK-1 has been given by Muller (458). Baird-Atomic Inc. has a phosphoroscope accessory, a strip scanner, and an adapter for solids for use with their SF-1 spectrofluorometer. This instrument has double grating monochromators for both excitation and emission which give it very high resolution and reduce stray light (40). The Perkin-Elmer Co. has discontinued their Model 236 spectrophotofluorometer and now offers the Hitachi Perkin-Elmer Model AIPF-2,1 fluorescence spectrophotometer. This instrument has a ratio-recording electronic system nith a xenon light source. Grating monochromators with an adjustable slit permit a resolution of 0.5 mp, -An accessory for phosphorescence measurements is also available (508). Carl Zeiss Inc. offers spectrofluorometers with a single prism monochromator for the exciting source or double prism monochromators with incandescent and deuterium bulb light sources (73Y). The Schoeffel Instrument Company's spectrofluorometer also u s ~ as prism on both the excitation and the emission with easily interchangeable tungsten or deuterium lamps (587). The Shimadzu spectrophotometer from Japan which has a fluorescence attachment is available from Ace Scientific Co. The instru-

ment has a littrow prism to analyze the emitted light and a xenon arc with a grating monochroniator which serves as the excitation source (60'7). A ratio filter fluorometer, with a phosphor coated sleeve on a mercury lamp source, is featured by the Beckman Instrument Co. The sample compartment of this instrument provideb for water cooling and a flow system accessory for automation (46). A nanosecond spectral source system is used in a fluorometer system of TRW Instruments to measure decay times of nanoseconds and microseconds (665). Practically all of the microscope firms noTv have fluorescence attachments. The Reichert Co. of Vienna, Austria, has a microphotometer available to measure the intensity of the fluorescence (548). The applications of this instrument have been described by Gabler (228). Additional fluorescence microscopes from abroad, available from representatives in the United States, are from Switzerland (709), England (678), and Japan (470). For the visual observation of chromatograms a new inexpensive cabinet is advertised (666). Iniproved spectrofluorometers have been developed in several research laboratories. A recording spectrofluorometer for the measurement of corrected fluorescence and phosphorescence excitation and emission spectra has been designed by Rosen and Edelman. Both right angle and frontal illumination may be used and a split excitation beam with an automatic gain control gives an independence from light source variatioiis (556). Boerresen (66) has described an instrument for obtaining true excitation spectra between 100' I< and 298' IUy~ectrophotometer for the measurement of total reflection fluorescence by which he studies concentrated solutions and thin films. The IKS-14 spectrophotometer has also been applied to the measurement of luminescence in partially VOL. 40, NO. 5, APRIL 1968

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transparent materials (24). Reduction of scattered light in fluorometers has bccn accomplished by placing a polarizer in the c~citingbeam. The exciting light is polarized in the direction in which the emitted light travels to the detector (117). Several new simple fluorometers have been developed for specific uses. A new filter fluorometer has been devised by Shcherbov, Plotnikova, and IGiptil’nyi (600)for measuring relative fluorescence intensity. Csanky (146) has produced a high sensitivity transistorized fluorometer for use in the determination of uranium. A simplified spectrofluorometer with a mercury arc lamp for an investigation of the flavins in the near ultraviolet and visible regions is described by Koziol (371). A fluorescence photometer designed for measuring the integral fluorescence intensity has been developed by Baumberg and Melikadze (45). \t7iersma and Lott have modified the fluorescence accessory for the Bausch and Lomb 505 and 600 spectrometers to allow automatic wavelength scanning and continuous f I o ~of the solution (708). The sensitivity of EF-311 fluorometer has been increased 7 times by an attachment with transistors (482). The solid sample accessory of the Aminco-Bowman spectrofluorometer is easily applied to front face optics for use mith opaque suspensions (715) and to studies on organic crystals from -170” to +160” c (57). Polarization is an important aspect of fluorometric analysis and two leading researchers in fluorescence with their associates have published improvements on polarization apparatus. Aurich and Lippert (23)have described the construction and performance of a fluorescence polarization fluorometer which they have developed. Weber and Bablouzian (695)also describe the construction and advantages of their fluorescence polarization spectrometer. The measurement of fluorescence on paper chromatograms and in thin layer chromatography has become an important part of fluorescence analysis. Bozhevol’nov and Xkolaeva (84)have described their apparatus for measuring intensities on a moving paper ribbon. Gordon (254) has coiistructed a quantitative scanning accessory for thin layer plates. I n air pollution studies nanogram quantities are measured on thin layer chroniatograrns (578). Horvath (301) also discusses quantitative scanning of fluorescent chromatography. Nybom (472)gives a method of marking to later identify the fluorescent colors of chromatograms on paper and Jackson (315) gives the exposures necessary for photographing the fluorescent colors. Some items on lamps are of interest in connection with fluorometers. Vurek (689) describes the construction and

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operation of a circuit to stabilize the fluorescent lamp for the Aminco Filter Fluorometer. Fluorescence microscopy with the quartz iodide lamp is discussed by Young (731). Muller (457) has an informative article on the resonance lamp detector in atomic absorption which also applies to atomic fluorescence. Isotope sources of low activity for fluorescent analysis are intriguing (484). Data on deuterium lamps is also of interest (309). The calibration and correction of excitation and emission spectra of the Aminco-Bowman spectrofluorometer has been clearly explained in an article by Chen (122). Eastman (191) describes a method using a light scattering solution of polystyrene to calibrate a spectrofluorometer and gives 22 references on this subject. Boerresen and Parker give important precautions to be observed in calibrating spectrofluorometers in the ultraviolet region (67). Methods for measurement of the absolute spectral sensitivity of phototubes are reviewed in detail by Lee and Seliger (395) and suggestions to eliminate errors are given. The influence of phosphoroscope design on the measured intelisity is discussed, and expressions are given for evaluating and designing phosphoroscopes in an article by O’Haver and Rinefordner (475). These authors also derive an espression for integrated luiniiiescciice intensity when using pulsing techniques (474). St. John, NcCarthy, and Winefordner (631) also discuss the application of the signal-to-noise theory in molecular lurninescence spectrometry. The fluorescent yield of uranyl salts under pulsed escitation has been reported (656). The cell position has been shown to be important in perpendicular type fluorometers (477). Pollack (521) has investigated the technique of tilting interface filters in a fluorometer and discusses the optical properties of interference filters with semitransparent silver films. A liquid filter, consisting of a saturated nickelcobalt sulfate misture in one container and a solution of 2,7-dimethy1-3,6-diazacyclohepta-1 ,&diene iodide in another efficiently transmits the 2537A line of mercury and excludes the remaining lines (90). McCarthy and Winefordner have an excellent general article on the selection of optimum conditions for spectrochemical methods and the quantum efficiency and decay timeof luminescent niolecules (436). St. John and Winefordner (630) evaluate time resolved phosphorimetry as a method of simultaneous analysis of two-component mixtures. INORGANIC

General. A general discussion of spectrofluorometry as a trace technique in inorganic analysis which includes t h e laws of fluorescence, ap-

paratus, reagents, procedures, a n d 31 references has been given by West (703, 704). Blyum and Shcherbov have suggested the term “threshold pensitivity” for fluorimetric determinations and have determined these values for In, Gal Te, Ag, Hg, Be, and Zr (63). The application of solid luminescent substances in analytical chemistry has been studied with 26 carrier compounds activated with 52 elements. The rare earth metals give the highest activation effect, and lo+% Cu, Nil and Co in luminescent zinc sulfide coatings may be determined semiquantitatively (298). 8-Mercaptoquinoline (thiooxine) RS a fluorometric ligand has been reported from three laboratories. Bankovskis, Bochans, and Ievins (43) have determined the absorption spectra and formulas of many thioosine metal complexes and state that the complexes with alkali metals, the alkaline earths, Zn, Cd, Hg, Sn, and Ga show a fluorescence. Anderson and Hercules (16) have determined the fluorescence spectra and quantum efficiencies of the thiooxine chelates with Cd and Zn. Davis (165) uses chloroform to extract the 8-thioquinolates of In, Gal Cd, and TI and shows their analytical possibilities. The fluorescent characteristics of the stable complexes of T1, Xg, P b , Pt, and Au with thiourea have been used to detect these elements by drop and microcrystallographic methods (530). Blyum and Pavlova (62) have extracted 24 complex and simple salts of Rhodamine B, Rhodamine 8,and crystal violet with benzene and benzene-acetone mixtures, and have determined their optical properties. These authors also measured the fluorescence intensities of the Rhodamine complexes. The higher sensitivity of the fluorescence methods, as compared to absorption, was ascribed to the lower blank values. The reactions of Eriochrome Blue Black R (C. I. 202) with about 25 metals have been tabulated and pK and ratio values of several complexes determined (615). -4review with 42 references on modern methods for the determination of impurities in reagents utilizes fluorimetry, polarography, and photometry (675). Some interesting results have been obtained on the detection of metals with Rhodamine B on paper chromatograms. For Au, Bi, Cd, and Hg a spray consisting of 0.025% Rhodamine B and 10% KBr in water gave red-violet spots in visible light which were deep violet under ultraviolet radiation (446). Aluminum. Two new fluorometric reagents have been developed for A1 ions. De A41biiiati(166) has shown that micro quantities of A1 from 0.004 to 0.44 pg/ml can be determined with 6- (2 -hydro,xy- 3- sulfo-5 - chlorophenylazo)-2-hydroxy- 1 - naphthalenesulfonate (Mordant Blue 9-‘2.1. 14855). White and his associates (705) have developed

N - salicylidene - 2 -amino - 3 -hydroxyfluorene (NSAHF) as a fluorometric reagent for A1 and indicate a sensitivity of 2 ppb in a water-ethanol solution. Dale, Jones, and Radley (158) show that in a DMF and acetic acid solution this reagent is quantitative from 0.02 to 2 pg of A1 in 25 ml. Radley’s group has made a thorough investigation of fluorometric reagents for A1 and Mg and state that P;S.UIF is the most sensitive reagent tested for ;il (157). Dagnall, Smith, and K e s t (147) have made a thorough study of salicylidene-o-aminophenol for the determination of submicrogram amounts of -41 and indicate a sensitivity as low as 0.2i ppb. This same reagent is reported to detect IO-?% A1 in germanium tetrachloride ( 8 6 ) , 10-670 in alkali metal salts and IO-?% in tin (398). I t is obvious that aluminum is contained in all reagents and distilled water in appreciable amounts (705) and should be removed before attempting determination of nanogram amounts. I n an opposite faqhion it has been shoL$n that organic impurities in pure NaCl decreased the sensitivity of the determination of A1 with salicylidene-o-aminophenol tenfold (34). Donaldson (178) uses Pontachrome BBR (Superchrome Blue Extra) to determine 2 ppb of A1 in natural rvater. The fluorescence of A I complex with 8-cluinolinol has been used for the determination of A1 in phosphoric acid (44), and in deodorants and antiperspirants (304). Ohnesorge (476) has studied the complex of A1 with 8-quinolinol in abqolute ethanol and indicates ratios of 2 : 1, 1 : 1, and 3: 1, depending on conditions. Palmer and Reynolds (487) have determined the ratios of -11, Fe, and Xi to the dihydroxyazo dye Solochrome J’iolet RS and report metal to dye ratios from 2:l to 4 : l on varying the pH from 4.5 to 6.5. Research on the A1 chelates with four natural or commercial flavonoids shows that morin is best of these to use for the fluorometric determination of Al. At the pH ranges of 3-4 or 5-6 morin gave a sensitivity of 5 ng per ml (250). Boron and Beryllium. Traces of boron in t h e order of 0.9 t o 3.2 X lO+y0 have been found in analytical grade sodium hydroxide with 4’-chloro2-hydroxy-4-niethosybenzophenone as a reagent in a concentrated sulfuric acid medium (416). This same reagent has been uped to determine boron in steel (449). The fluorescence of the boron complex with l-(o-arsenophenylazo)-2hydroxy-3,B-naphthalenedisulfonicacid also in concentrated sulfuric acid has been used to determine 3 X lo-*% B in silicon tetrachloride (546). A unique method for the determination of boron extracts the complex of boron with orthohydroxybenzoic acid and Rhodamine 6G, n i t h benzene (37). I n this method iron (11) is used to complex the excess

hydroxybenzoic acid. No new reagent, have been proposed for beryllium in the past two years. A review of the analytical methods for I3e has been published (445) and applications of the morin method to special cases such as the determination in submicrogram amounts (619) ancl in paper pulp analysis (451) have been given. Calcium, Magnesium, and Lithium. Ashtoii (22) has shown that tetracycline serves as a good fluorescent indicator for the compleximetric microdetermination of Group I1 cations. Directions are given for the use of this indicator in titration of Ca, Ba, Sr, and Mg with disodium EDTA. Tetracycline gives a yellowish green fluorescence with a max4mum at pH 10 in the presence of these metal ions. Escarrilla (205) uses calcein blue as an indicator for the titration of Ca, l l g , and Fe (11). Palyi (488) reconnnends luminol as a fluorescent indicator in the titration of l l g . The determination of serum Ca with calcein indicator has been automated (288). Dagnall, Smith, and West have studied the magnesium complexes with quadridentate azomethines (150) and conclude that N,N’-bis(salicylidene)-2,3-diaminobenzofuran is an excellent reagent for Mg as low as 2 ppb (149). Bozhevol’nov after a study of chlorazo compounds shows that 2 ng of M g per ml may be determined with the reagent 1- (2- hydroxy- 3-sulfo-5-chloro-phenylazo)-2’-hydroxynaphthalene (81). The use of 8-quinolinol-5-sulfonic acid for the fluorometric determination of Mg has been the subject of several investigations. Patrovsky (505) providcs for the removal of Ca and also for the analysis in the presence of Ca (506). Bishop gives the formation constants of Mg and also of 7 other ions with 8-quinolinol (69). Clark and How (130) improve the 8-quinolinol method by the removal of Ca and phosphate. Klein and Oklander (351) use 8-quinolinol-5-sulfonic acid for an automated method for N g , and Breen and Marshall (95) use o,o’dihydrosyazobenzene for an automated method for Rlg in serum and urine. Another procedure uses an ion exchange strip in the automated determination of Mg (225). A new fluorometric reagent for lithium, dibenzothiazolylniethane,has been proposed by Pitts and Ryan (517). This reagent will determine 10 pg of Li in 10 ml of solution and has been used to analyze for the Li in alkali metal salts, Zinc also gives a strong fluorescence with this reagent. Cadmium and Zinc. Bozhevol’nov has shown t h a t both Cd and Zn in amounts of 10 ng in 1 ml may be determined with t h e reagent 8-(ptoluenesulfonilylamino) quinoline (81). This same author with his associates ( 5 9 4 , in an extensive study of the 8-(arylsulfonamido) quinolines as rea-

gents for Zn and Cd show that, of the compounds studied, 8-(phenylsulfonamido) quinoline, 8-(p-tolylsulfonamido)quinoline, and 8-(niesitj lsulfonamido) quinoline were the most seniitive for Zn and Cd. The second of these has been used to determine 0.1 pg of Zn per ml of the choroform extract of the complex (595). Jensen and Pflaum (319) have shown that picolinealdehyde-2quinolylhydrazone may be used to determine 26 ng of zinc. I n this case the complex is extracted into CC14where the fluorescence is measured. Ryan, Pitts, and Cassidy (564) have made a study of the fluorometric determination of Cd in 1 to 250 ppb with 8-quinolinol5-sulfonic acid and have listed the effect of many other ions. Atomic fluorescence has been knom 11 for several years to be sensitive to 0.1 ppb of Zn and has now been shown to detect traces of Cd (155) t o about 1 x 10-9Jf. Chromium, Copper, and Gold. T h e fluoro- and chloro-complexes of chromium have been shown t o produce fluorescence a t t h e temperature of liquid air (585) and present the possibility of a n analytical method, A determination of copper (11) in the range of 1 to 6 ppb (39) depends upon the fluorescence of the complex of Cu(I1) a i t h phenanthroline and the dye Rose Bengal. The complex is extracted into chloroform diluted n i t h ammoniacal acetone. Another trace method for copper has been used t o determine 5 ng of Cu in alkali metal chlorides. This method is dependent on the formation of a fluorescent Cu complex with p-di-

methyl-benzylidene-beiizoylaminoacetic acid (379). I3randt and Jones (92)have reported on a quenching method with a sensitivity of 10 ng of Cu(1) per ml, which depends on the quenching of the fluorescence of 2,gj-dimethy1-4,7-diphenyl-1 10-phenanthrolin~. Four groups of authors have published methods for the deterniination of traces of du(1II) by the eytlaction of Rhodamine-.1u complexes into benzene. Butyl Rhodamine C (560), Rhodamine S (BSS), and Rhodamine B (642) were used and in the last case a sensitivity of 0.5 pg of h u per ml was reported. A thorough study of the determination of Au in ores with Rhodamine T3 has been made by Xarinenko and May (418) who recommend the extraction of the AuRhodamine I3 compleu from 0 4.V HC1 with isopropyl ether for the fluorometric measurement. -4s Ion. a. 3 ppb in the ore can be determined by this procedure. Iridium, Osmium, Cobalt, Nickel, and Iron. Wunschel and Ohnesorge (726) have made an evtenrive ptudy of the luminescence of the Tr(IT1) chelates with 2,2’-bipyridine and with 1,lOphenanthroline and find that both of these chelates fluoresce in dimethylformamide solutions. Absorption and fluorescence emission spectra of the ~

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fluorescent chelates and quantum yields are given. Burchett (101) has developed 4,6-bis(methylthio-3-aminopyrimidine) as a reagent for Os and detect.. 50 ppb. Another determination of Os in about this same sensitivity range involves the use of OsOl as a catalytic agent in the reduction of Ce(IT’) to Ce(II1). The amount of Ce(II1) is measured by its fluorescence (345). X kinetic method of Bozhevo1’nov aiid Kreingol’d (83) will determine 0.5 ng of Co in 5 ml. I n this method salicylfluorene [9-(hydroxypheny1)-2,6,7’-trihydroxyfluorone] is oxidized by hydrogen peroxide with Co as a catalyst. Kickel may be determined by titration with S a 2 E D T B using bisglycinemethylene-dichlorofluorescein as an indicator (421). Escarrilla (205) determines iron in a similar EDTA titration with calcein blue as an indicator in a solution containing formaldehyde, hydrogen peroxide, and ethanol. Babko and Kalinichenko (33) have found that the presence of triethylenetetramine in the luminol-hydrogen peroxide system permits the detection of 0.08 ng of iron per ml. Osipov (479) recommends a determination of iron which involves the decrease in the fluoresceiice of the uranium fluoride bead. Antimony, Bismuth, Gallium, Germanium, and Lead. Bozhevol’nov and Solor’ev use t h e low temperature fluorescence of t h e chlorides in HCl solution to determine Pb (89, 623), Bi (623),aiid Sb ( 8 7 ) . The fluorescence of the lead chloride complex may be observed a t -7’0’ C or at room temperature; the fluoresceiice of the Bi chloride compleh is not visible a t these temperatules and is measured only at -196’ C ; the fluorescence of the Sb complex is measured a t -70’ C. Gallium gives a strong fluorescence n i t h 2,2’,4’-trihydroxy-5-chloroazobenzene-3-sulfonic acid nhich may be used to determine 1 iig of Ga in 1 ml (81). If the Ga complex a i t h this reagent is extracted with 71-hexanol, 4 ng per liter may be determined (650). Holzbecher (297) extracts the Ga complev of salicylaldehydeacethydrazone with isoamylalcohol from saturated sodium perchlorate to determine 7’0 ng of Ga per ml. Elsheimer (200)has described an indirect determination of Ga accurate to O.1Y0 M hich depends on the addition of EDTA to the sample and back titration with copper in the presence of calcein blue as an indicator. The composition of the Rhodamine B metal complexes have been determined bj7 means of chlorine analysis and found to be of the general type (RH)SbCls,(RH)TlCh, etc. (308). The authois of a report on a new colorimetric reagent (Rezarson) for Ge state that it may also be used fluorometrically. They give a colorimetric sensitivity of 3 pg in 5 ml but no fluorometric data are given (410). 120 R

ANALYTICAL CHEMISTRY

Lanthanides. Several articles of general interest on t h e fluorescence and charge transfer complexes of the lanthanides have appeared. Crosby (144) has published a review with 106 references on the luminescent organic complexes of the rare earths. RlcCarthy and \T7inefordner (434) have discussed intermolecular energy transfer as a means of analysis and sensitization of rare earth emission. Short and Parker (610) also discuss the fluorescence of charge transfer complexes in solution. Heller (279) has shown that the substitution of D 2 0 for H20 enhances the fluorescence of the lanthanides. Eisentraut and Sievers (197) have prepared and determined the composition and spectral characteristics of 15 lanthanides with 2,2,6,6-tetramethyl3,5-heptanedione H. These are stable volatile compounds in a ratio of 3 ligand units to one metal ion and in several cases they were found to produce an intense fluorescence. Kononenko and his associates have published a series of papers on their research on complexes of the lanthanides with 1-aryl-3-methyl1 10-phenanpyrazol-5-ones (651j ; throline-dibenzoylmethane (441); 1 10phenanthrolinethenoyltrifluoroa ce t o n e (364); and with acetylacetone and 1 , l O phenanthroline or 2,2’-dipyridyl (361). This group employed the above reagents in the analysis of tributyl phosphate estracts and reported the sensitivity in per cent as: Sm& 0.5; Eu& 0.008; TbzOa, 0.05; n3-203,0.2 (443). Kreher, in an article with 33 references on the fluorescence characteristics of the rare earths, s h o w that while the short wave absorption spectrum depends on the ligand, the fluoresceiice emission depends on the metal ion (378). Stanley, Kinneberg, and Varga (626) have reported their results on the spectrofluorometric analysis of rare earth chelates of thenoyltrifluoroacetone, benzoylacetone, and dibenzoylmethane by computer spectrum stripping techniques and have shown that this method is feasible for analysis. These authors showed that an emission monochromator and detector sensitive in the near infrared were necessary to detect the intense fluorescence lines of Pr, S d , Dy, KO,Er, and Tb. Alberti and Nassucci have published extensively on the determination of the lanthanides in tungstate and oxalate solutions and show that these media give far greater sensitivity than HC1 solutions for Sm, Dy, Eu, Tb, and Ce (6). The fluorescence of the complexes of -41, Sc, Y, La, Lu, and Gd a t low temperature is discussed, with 15 references, by Bozhevol’nov and Solov’ev as a sensitive method for the determination of inorganic impurities (88). Europium and terbium have received more attention in fluorometric analysis than the other lanthanides. Sinha ~

~

(616) has three papers dealing with 2,2’-dipyridyl complexes of rare earths and s h o w the effect of ligand substitution on the fluorescence intensities of the Eu and T b chelates. I3allard and Edwards (42) state that of the trivalent rare earths only the E u and Sm comple\es, with thenoyltrifluoroacetone, fluoresce strongly in solution at room temperature. This reagent has been used to determine 5 X 10-470 E u in La203(469) and the stability constants a t several pH levels have been determined (384). The fluorescence of other fluorinated p-diketones has been the subject of a research by Charles and Riedel (112). The dibenzoyl and 1,lOphenanthroline complexes of E u have been studied (106) and found useful in analysis (524). Carbonate solutions have also been found satisfactory in the determination of T b and E u by spectrofluoresceiice (6.41). Traces of Tb can satisfactorily be determined in oxalate solution (?) or with sulfosalicylic acid complexes (113, 148). The phenylsalicylate complex (362) with T b exhibits a bright green fluorescence in alkaline solution. The antipyrenesalicylate complex may be extracted u i t h benzene (652) for the determination of Tb. There is a possibility that Tb sensitizes the fluorescence of E u in some cases (525). A procedure for the determination of Eu aiid T b in mineral concentrates by mean3 of fluorescence spectroscopy has been outlined (339). Solvent and temperature effects on the intensity of the fluorescence emission of Eu-p-diketonates have been found to be of appreciable importance (212). Samarium and E u may be determined quantitatively by the fluorescence of their complexes n ith thenoyltrifluoroacetone or by the fluorescence of the suspension in organic bases as diphenylguanidine (442). Samarium in the concentration of 5 X in CeOz has been determined by the fluorescence of the solid pellet phosphor made by grinding the mixture n ith lead sulfate (207).

Holzbecher (297) has shown that the fluorescence of the Sc complex with salicylaldehydeacetylhydrazone extracted with isoamyl alcohol will detect 50 ng of Sc per nil. The salicylaldehyde semicarbazoiie complex is also used for the determination of traces of Sc (344). Petronio and Ohnesorge (612) have made an absorption and spectrofluoresceiice study of the 8-quinolinol coniple.; with Sc aiid have shown it to be a 3 1 ratio in absolute alcohol. Pyrazolone derivatives have been found to form compleses 15 ith dysprosium which are used to determine 0.005% of the o d e of this element in other rare earth oxides (663). T\$o of these pyrazolones comple.; with thulium and shoir a sensitivity for the detection of about 7.5 to 10 pg in 10 ml (363).

for measurement of the fluorescence of Shigematsu, Nishikawa, and Hirakai the U-fluoride-carbonate pellet. This (604) have shown that yttrium can be instrument focuses the light from a high determined fluorometrically by means of pressure mercury vapor lamp onto the the chloroform extract of its complex pellet by means of a mirror and the with 5,7-dichloro-8-quinolinol. Phefluorescence emission is measured with a nylsalicylate is also used for the fluorophotomultiplier tube (567). A commetric determination of yttrium after a parison of the determination of U by the paper chromatography procedure (343). fluorescent bead method has been made Cerium may be determined in the presto the hydrogen peroxide and thioence of other lanthanides by its emiscyanate colorimetric methods. The sion a t 350 mp provided the sample is peroxide method is deemed suitable for converted to chlorides (525). 0.1 to 0.5% of USOSwith an error of Vanadium, Molybdenum, and about f3%, the thiocyanate from Tungsten. Bognar and Jellinek (68) 0.005 to 1% with an error of about have devised a n unusual type of inf4Y0 (667). Bruce and Ashley describe direct ultraniicro fluorescence dethe collection and determination of termination of vanadium. I n this trace amounts of U on cellulose phosmethod vanadium(V) is treated with phate. The excitation and fluorescence a bromate-bromide ascorbic acid mixspectra were found similar to those of ture along with the dye Trypan Red (p,p'-diaminodiphenyl-vi-sulfonic acid phosphate solutions (99). The effect of diazo- bis-2-naphthylamine-3,6-sulfonic temperature on the spectra of solutions of uranyl nitrate and acetate in glycacid). This dye is oxidized by erol, ethanol, and acetone has been bromine to yield a product that fluoresces an intense blue. Another micro determined from - 186' to 20' C. More complex spectra are observed in organic method (35) for the determination of than in inorganic solutions (687). vanadium depends on the quenching of Uranyl nitrate 4 X 10-7 M may be the chemiluminescence of the luminoldetermined in sodium nitrate or sodium H202-Co(III) system by vanadium(V). carbonate solutions by its fluorescence Carminic acid serves as a fluorometric spectra (176). reagent for the determination of 0.1 to Nonmetals and pH Indicators. A 0.9 ppm of 11o(VI) a t a pH of 5.2 and simplified instrument has been defor 0.4 ppm of W(V1) a t a pH of 4.6 signed for the determination of fluoride (347). h wide range of ions was in the air with a tape containing investigated with this reagent and a Mg-8-quinolinolate and a single procedure for thfidetermination of 110 photomultiplier tube to receive the in steel down to 0.0170 is given (346). light pulses (649). An interesting senBottei and Trusk (75) have shown that sitive method for the determination of after an ion exchange separation ozone which is not affected by YOz or flavonol may be used as a fluorometric SO2 has been the subject of three reports. reagent for tungsten. The reaction mixture is an ethanol soluThorium and Zirconium. Flavonol, tion of gallic acid and Rhodamine I3. the long established reagent for Zr, The ozone is thought to convert the has now been applied to the fluogallic acid to an activated intermediate rescent determination of thorium in which in turn produces a chemilumi25% ethanol solution a t a pH of 2.2 nescence of the dye (53, 541, 628). A (79). RJorin continues to be used as a novel enzymatic method has been dereagent for the determination of Zr in vised for the analysis of inorganic phossea water (606), in trichlorosilane (82), phate which is sensitive to 0.02 nanoand in an automated method (617). mole. The phosphate is mixed with I n the automated method poor results on duplication were experienced. It is glycogen, phosphatase and TP?UT+,the highly fluorescent T P N H results (589). unfortunate that flavonol Ivas not used Land and Edmonds have studied the in this as well as the other cases for it quenching of the fluorescence of the was shown 14 years ago to be an excelchelates of Al, Gal and Zr, with morin, lent reagent for the determination of Zr. quercetin, and flavonol with phosphate A chemiluminescence method for the ions and conclude that of these comanalysis of Zr depends upon its quenchpounds the quenching of -4l-morin fluoing of the luminol-copper-hydrogen rescence provides the most satisfactory peroxide system (SO). trace method for phosphate determinaUranium. Samsoni has made a tion (388). The fluorometric method thorough study of the factors affectwith 3,3'-diaminobenzidine has been ing the accuracy and sensitivity of the used to determine traces of Se in hydrouranium determination with the XaFchloric acid (85) and in biological maN a K C 0 3 fusion. The fusion temperaterials (643). Watkinson prefers 2,3ture, rate of cooling, spurious rediaminonaphthalene for submicrogram flections of light, and the nature of amount of Se (694). the filter are some of the factors covered A number of new p H indicators for in this work (566). Osipov has presented curve5 for the quenching effect of the p H region of 13.3 to 14 have been iron in the U determination (480). A produced which are derivatives of bis(triazinylamino) stilbenes and contain reflection fluorometer has been designed

hydroxyethylamino, carboxymethylamino, and bis(carboxymethy1)amino groups in the triazine ring (389, 644). Luminol is recommended as a pH indicator with an effective range of 7.1 to 8.2 (203). Three reagents are recommended by authors for the determination of sulfide. Wronski (721) has devised a simple arrangement for the titration of sulfide with tetra-(acetoxymercuri) fluorescein acetate. This same reagent is used in the detection of sulfur compounds (274) and in the determination of lO-7yO sulfide in trichlorosilane and lo-*% in water (597). Bishop (60) uses the Cd complex of 8-quinolinol-5-sulfonic acid at p H 6 to titrate sulfide. Another fluorescent method for sulfide involves the precipitation of sulfide with cupric chlorate, filtration, and titration of the excess copper with di-sodium EDTA with bis{ [(carboxymethyl)-amino]methyl} dichlorofluorescein as a fluorochromic indicator (52). A fluorescence method, for the analysis of traces of sulfate, of Guyon and Lorah involves the addition of sulfate to a thorium solution and then the addition of morin to determine the T h not held by the sulfate. Ions which complex or precipitate with T h or form fluorescent products with morin interfere in this determination (266). The measurement of the amount of Ce(III), formed on the reduction of Ce(IV), by spectrofluorescence, has been shown to provide a method for the determination of the amount of reducing agent involved. Oxalate, As (111), and Fe(I1) have been measured by this means (345). Traces of moisture in organic compounds may be detected by means of 4-chloro-2-sulfobenzalacetophenone. This compound exhibits an intense green fluorescence which disappears if the water content is over 0.00570 (37'7). Eisenbrand and Hett (195) report that a previous statement that distilled water had a slight blue fluorescence is in error and was probably caused by light scatter and Raman effects. Atomic Fluorescence. Only relatively few references on atomic fluorescence will be given here for representatives of the two leading groups in this field, Winefordner and Mansfield (712) and West (702), have published recent reviews on the subject. Certainly this is one of our most sensitive means of analysis. Both Cd (155) and Zn (415) can be determined in the order of 0.1 ppb and several other elements also approach this sensitivity as methods for this technique improve. Dinnin and Helz (176) describe a demountable cathode lamp as an excitation source for atomic fluorescence and Dinnin gives a series of results with this source (174). Results with a xenon arc as a continuous source show less sensitivity VOL. 40, NO. 5 , APRIL 1968

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than other sources (414). Ellis and Demers show the use of a hydrogen-air flame in atomic fluorescence (199). Armentrout uses a hollow cathode Ni lamp and a hydrogen-air flame in the determination of Ni (21). West and his associates (153-1 65) have a series of informative articles on atomic fluorescence spectroscopy; one of these (152) discusses the determination of Se with a sensitivity of 0.2 ppm and Te of 0.12 ppm; another (151) deals with the determination of Sb to 0.05 ppm. Winefordner and his group have published a series of general articles dealing with the selection of optimum conditions for spectrochemical methods including atomic fluorescence (713, 495, 436). Acid concentration is an important factor in the solutions used in atomic fluorescence (93). Goodfellow (263) has a 19-page article on factors in atomic fluorescence and has also experimented on interelement effects (252). Winefordner and his associates have derived expressions for the calculation of limiting detectable concentrations in atomic fluorescence flame spectrometry (710) and also for quantum and power efficiencies (433). Chemiluminescence. A 250-page book on Chemiluminescent Analysis (29) gives a good review of analytical applications of this subject. The authors of this book also have published a 12page article on chemiluminescent methods of analysis (36). Vasil'ev (673) has given a review of chemiluminescence in solution, with 158 references. Haas (267) has also an excellent article on chemiluminescence in solutions; it includes history, methodology, mechanisms, analytical applications, and 76 references. Two articles on the theory and kinetics of Chemiluminescent reactions are of interest (337, 490). Intensive research projects of several commercial companies have uncovered many new chemiluminescent systems. Much of this work has been directed toward the production of light by oxygen activated systems to be used for marking exits in case of electric failures, to illuminate aircraft in case of accident, and other commercial applications. Reports from the Monsanto Research Laboratories (49, 183, 184) state that over 175 organic compounds were investigated in the Me2SO-tert-BuOK-Oz systems. Almost every class of compound studied gave a t least a weak emission. The indoles were the most promising class and skatole provided the brightest system. Patent applications also list many compounds studied a t the American Cyanamid Co. laboratories for chemiluminescence (538, 539). Apparatus designed especially for chemiluminescent measurements has been described by Roberts and Hirt (549), by Van Pul (672), and others (674). The chemiluminescent

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reaction system of luminol-hydrogen peroxide-copper has been studied with reference to the optimum pH and rate of decomposition of H202 (18). Siloxene is reported to be a good reagent for the analysis of traces of Mn (0.1 ml of 0.0004M MnSOd) after oxidizing the M n to permanganate with persulfate (31). A 19-page report on redox reactions by Erdey and Buzas (202) shows that it is possible to titrate oxidizing ions with hydrogen peroxide with lucigenin (N,N'dimethyl-9,9'-biacridinium nitrate) as an indicator. The chemiluminescence of lucigenin with and without metal ions present has been studied, and the concentration of cation required to double the emission intensity has been determined for Co, Ag, Pb, Bi, Cr, Cu, hln, Ni, and Ce (32). The kinetics of the chemiluminescence decay of lucigenin and of four of its analogs have been studied and the results are depicted in 70 exponential curves (94). A theoretical study of chemiluminescence generated a t electrode surfaces is the subject of a paper by Feldberg (211). The chemiluminescence spectrum of rubrene on electrolysis has been shown to be identical with its fluorescence spectrum as is generally true with these two types of luminescence (283). Kazakov and Lapshin (333) have demonstrated that the lanthanides on electrolysis in sulfuric acid solution exhibit both visible and ultraviolet chemiluminescence. The chemiluminescence of humic acid is evidenced when this substance is oxidized with hydrogen peroxide in 0.5% ethanol which is 0.125M with sodium carbonate (619). Weller and Zachariasse (701) have shown that perylene and seven other aromatic anions produce chemiluminescence on mixing dimethoxyethane solutions of their radical anions with a suspension of Wurster's Blue perchlorate in the same solvent. An interesting case of chemiluminescence is produced on the reduction of germanium tetrachloride with potassium vapor (648). Several applications of chemiluminescence in analysis are listed above in the sections on iron, ozone and zirconium. ORGANIC AND BIOLOGICAL

Because of the rapid increase in papers on luminescence methods in organic and biological chemistry, greater selectivity in coverage has been necessary this year than in the previous reviews. Several broad surveys have been published, such as an extensive one on fluorescence and phosphorescence of organic molecules by Wehry and Rogers (698) and another by Wehry (260), two on fluorometry in pharmaceutical analysis (76, 218), and one on the three types of long lived fluorescence found in organic compounds (393). Winefordner

has given a detailed discussion of the analytical uses of phosphorescence for determining alkaloids, aromatic hydrocarbons, pesticide metabolites, and physiologically active amines and other drugs (711). A later chapter on this subject is given by McCarthy and Winefordner (260). There have also been reviews on quasilinear ffuorescence spectra (317), the theory and applications of phosphorescence in organic chemistry (69)' and fluorescence methods in biomedical research (671), clinical pathology (514, and identifying inborn errors of metabolism, with detailed methods for porphyrins, sugars, and amino acids and their metabolites (305). Aromatic Hydrocarbons and Heterocycles. Berlman has published a handbook of fluorescence spectra of aromatic molecules (61). The changes in the fluorescence of aromatic hydrocarbons caused by pi-electron acceptors such as tetracyanoethylene have been studied (584). Parker has described the precautions necessary in trace analysis for aromatic hydrocarbons, with examples such as determining 1 ppm of anthracene in zone-refined phenanthrene (494). Research has continued on delayed fluorescence in compounds such as perylene (491, 492) and pyrene (189). Benzo(a)pyrene has remained a center of attention, because of its carcinogenic nature. Luminescence procedures are reported for it in cigarette smoke (548),with a large excess of perylene as an internal standard ( 1 6 4 , in airborne dust (318, 485, 611)) in highly purified industrial paraffins using the 4030A line (596), and down to the 10 ng/ml range by taking the ratio of the 4030A and the 4086X lines in the quasilinear spectrum a t 77" K (614). Several other workers abroad have used the fine structure luminescence analyses for benzo(a)pyrene (284, 403, 468) in various industrial and natural products (338),and down to 25 ng/ml in benzene solution even in the presence of 3,4benzofluoranthene and 1,2-benzopyrene as contaminants (431). Howard et al. have described a method for determining benzo(a)pyrene in the 1 ppb range (309, 303), after separation of the polycyclic aromatic fraction from smoked foods with the aid of a 2,2,4trimethylpentane extraction; this gives greater sensitivity than an earlier method which used hexane, dimethyl sulphoxide, and benzene extractions (232). Phosphorescence spectra a t 77" K are rapidly coming into use for analytical purposes. Excitation and emission curves have been published for 40 polynuclear aromatic hydrocarbons and halogen derivatives (417 ) . The intensity distribution in phosphorescence spectra has been studied for coronene and triphenylene in carbon tetrachloride

solid solution with solute-solvent interaction (105), for o-xylene, m-xylene, and mesitylene in cyclohexane solution and for diphenyl in petroleum ether (354), and for naphthalene and diphenyl at 20' K (646). Hood and Winefordner have given the excitation and emission peaks for 12 aromatic hydrocarbons such as anthracene and benzo(a)pyrene, and described an attempt to increase the sensitivity of phosphorimetric analysis by the external heavy-atom effect: adding ethyl iodide to increase the probability of singlet-triplet transitions (299). Zander used the same principle in trying to intensify the phosphorescence of several aza-hydrocarbons in EPA solvent by the addition of methyl iodide (733). I n both of these studies, the increase in sensitivity achieved was not very large. Phosphorescence analysis has been applied also to the identification of contaminants in technical pyrene and the determination of impurities in purified anthracene (737) as well as to polynuclear hydrocarbon and heterocyclic constituents of coal tar (734, 736). Luminescence spectra at room temperature and 77" K, and their analytical uses, have been described for many other aromatic hydrocarbons, including fluoranthene (670), acenaphthene in paraffinic solvents (413 ) , and phenanthrene and anthracene in methyl cyclohexane solution (466). Quasilinear spectra have served in a control method for measuring the purity of pentacene and coronene (ZO), in determining low concentrations of dibenz(a,h)anthracene (367), and in the determination of 7,12-dimethylbenz(a)anthracene in complex mixtures (163). Dual fluorescence spectra of biphenylene (from the second excited singlet as well as the first) have been reported (56) and refuted (459). The complex of pyrene with dimethylaniline has been studied by its strong fluorescence in nonpolar solvents (426) and the spectra of seven sandwich dimers of substituted anthracenes have been discussed in terms of their electronic transitions (109). In six perfluorinated and perchlorinated aromatic compounds (such as octachloronaphthalene or decafluorobiphenyl), fluorescence and phosphorescence yields and the decay times were studied a t 77" K in ethanol-ether solution (204); reduced quantum yields were observed, and the red shift was greater for the chloro derivatives. In combining gas chromatography with luminescence analysis for petroleum fractions, Drushel and Sommers found that phosphorescence gave more information and sensitivity than fluorescence (188), and were able to observe both pyridine and quinoline luminescence by changing the excitation wavelength. Other luminescence studies reported include pyrazine (405), phena-

zine derivatives (425),indoles (691,707), and 1-indanone (725). Sawicki and associates have identified various polynuclear hydrocarbons (576) and azaheterocyclics (579) in particulate air pollutants, by means of thin layer chromatography in conjunction with luminescence characteristics. Oxygenated Molecules. An excellent paper on organic functionalgroup fluorometry has been written by Pesez and Bartos (509), giving detailed procedures for 1,2-diols, aldehydes with an a-methylene group, hexitols, hexuronic acids, a,p-dihydroxyacids, 17ketosteroids, aliphatic aldehydes, phenols, 2-deoxysugars, primary alkylamines, and a-aminoalcohols, and several other classes, mostly a t concentrations around 0.1 pglml. One of their principal techniques is to convert the reactive grouping into formaldehyde by oxidation with sodium periodate, then condense the formaldehyde with acetylacetone in the presence of ammonia to yield a diacetyldihydrolutidine with strong yellow-green fluorescence. Enzymatic fluorescence methods have been used to analyze for ethanol in blood using alcohol dehydrogenase (447),and also for glycerol using coupled glycerokinase-glycerophosphate dehydrogenase (390); in both cases, the amount of reduced nicotinamide adenine dinucleotide (NADH, formerly called D P K H ) produced from the substrate is measured fluorometrically. Another procedure either for glycerol or for triglycerides in serum, now completely automated (336),is based on periodate oxidation to formaldehyde, which is condensed with acetylacetone and ammonia to give a highly fluorescent lutidine derivative. Lipids and lipoids have been estimated in vivo and in vitro by fluorescence microscopy, with the help of fluorochromes such as Nile Blue or Neutral Red to induce additional luminescence ( 8 ) . Lecithin has been determined in vitro and in carp yolk grains by its specific yellow-green fluorescence after staining with 3formyl-10-methyl-phenothiazine (17 7 ) . The radiothermoluminescent behavior of oleic acid has been reported (25). Other papers have described the effect of pH on the fluorescence of aqueous p-hydroxydiphenyl (691), low temperature spectra of some phenols (396), and the absorption and fluorescence peaks of four isomeric hydroxyskatoles (276). Sawicki's group has described fluorometric procedures, related to air pollutants, for succinaldehyde by condensation with o-phenylene-diamine (577), for a number of aldehyde and ketone 4-nitrophenyl-hydrazones by their luminescence a t room temperature and at 77" K (571), for 9-acridanone (570) and 7H-benz(de)anthracene-'?-one (572) using thin layer chromatography, for three-carbon fragments (containing the

carbonyl group) with anthrone reagent (673), and for cyclohexane-1,4-dione, by condensation with o-phthalaldehyde to form pentacenequinone (574). Reports are available on the absorption and luminescence spectra of the acenequinone family from 1,4-naphthoquinone and anthraquinone up through heptacene-7,16-quinone (735),as well as various alkyl- and arylanthraquinones (598). The phosphorescence spectra of several carbonyl compounds have been noted ( 4 ) , and the 2-hydroxy-5methoxybenzohydrazides and 5-methoxy-3-nitrosalicylhydrazides have been proposed as fluorescent derivatives of carbonyl compounds (172). Acrolein can be determined by fluorometry after reaction with resorcinol in the presence of any of a number of amines ( 5 ) ,which suggests the possibility of sensitive methods also for allylamine, benzylamine, spermine, etc. Carbohydrates have been the subject of several fluorometric methods. Nanogram quantities of inulin have been determined, and glucose distinguished from fructose, by Vurek and Pegram (690) using Adachi's earlier method of reaction with dimedon and phosphoric acid. Ketoses when heated with zirconium oxychloride solution form an intensely fluorescent 1 :1 chelate (660). A number of hexoses and pentoses were determined in nanogram amounts by dehydration in concentrated hydrochloric acid to give furfurals, which upon condensation with resorcinol yield intense fluorescence in alkaline solution (555); the nature of the fluorescent substance produced from fructose by acid was investigated (729). Aldose, ketose, pentose, methylpentose, and disaccharide analysis by fluorogenic condensation with aniline or @-naphthylaminein butanol solution containing phthalic acid is suitable for the microgram range (406). Glucose was determined by titration of excess cupric ion using a metallofluorochromic indicator (42S), and mixtures of hexoses have been analyzed by automated fluorometric column chromatography (696). Glycogen can be measured with a sensitivity of 50 ng by enzymic conversion to 6phospho-gluconolactone plus NADPH, followed by fluorometry of the latter (499). The same principle of measuring the fluorescence of the pyridine nucleotide coenzymes reduced as a result of coupled enzymatic reactions has been employed by Lowry's group (248) for determining the Krebs cycle intermediates glucose6-phosphate, citrate, isocitrate, oxalacetate, a-ketoglutarate, pyruvate, succinate, malate, and fumarate. Similar methods were reported by others for pyruvate with automation (219), glucose-6-phosphate in liver (473), acetoacetate or 6-hydroxy-butyrate in blood VOL. 40, NO. 5, APRIL 1968

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(?SO), and these three plus lactate in plasma (19). Residues of terephthalic acid down to 0.1 ppm in chicken tissues have been determined by nitration, reduction to the amino derivative, and fluorometry of the latter in butanol solution (237). Variations caused by temperature have been reported in the phosphorescence spectra of phthalic acid (349) and terephthalic acid (350). After preliminary separations and paper chromatography, salicylic and acetylsalicylic acids in blood mere quantitated by their fluorescence a t 415 mp (388). Sebacic acid can be determined fluorometrically at 440 mp by condensation with resorcinol in hot concentrated sulfuric acid, but other dibasic acids interfere in this method (258). Seven flavones and 12 flavonols were analyzed qualitatively and quantitatively by the fluorescence of their aluminum chelates (394). Karingin, naringenin rutinoside, and related flavanone glycosides in grapefruit juice were isolated by chromatography and quantitated fluorometrically (870). The intense fluorescence of the product 7 hydroyy-coumarin (umbelliferone) provided a means to follow the hydroxylation of coumarin by liver microsomes (143).

Fluorometric procedures have been described for such urinary metabolites as 5-hydro\;)-indoleacetic acid, in 3N HC1 after Sephadex separations (137), and 3,4-dihydroxyphenylaceticand 3,4dihydrosy-mandelic acids after condensation with ethylenediamine (186). hlkosycinchoninic acids and ~ o i n erelated quinoline carboxylic acid derivatives show intense fluorescence which can be used for quantitation of microgram amounts in methanol or chloroform solution (fO3). Vitamins. The intrinsic fluorescence of vitamin A at 490 mp in cyclohexane solution can be used for its determination at normal fasting levels in 0.1 ml of blood (325). A1though it is not very specific, the fluorescence a t 475 mp produced by heating vitamin D2 or DS in ethanolic sulfuric acid (49'7) may serve as a quantitative method under some circumstances. Of the vitamin E compounds, only the free form of tocopherol shows fliiorescence a t 340 mp (n-ith 295 mp excitation) but any tocopheryl esters can be reduced to the free form with lithium aluminum hydride, in a recent microfluorometric method for total tocopherols in serum (273). A microfluorometric assay for vitamin C is based on Oxidation to the dehydro form, followed by Condensation with o-phenylenediamine to form a quinosaline fluorescing a t 430 mp (1'70, 171); the method does not distinguish between l-ascorbic and disoascorbic acids, but these can be sep124 R

ANALYTICAL CHEMISTRY

arated by chromatography on glass fiber paper (697). Riboflavin determinations by the lumiflavin fluorescence method have been reported for urine (448), foods (107, 108), and foods after liberation of the riboflavin from combined form in nucleotides, by heating a t 160' in 50% aqueous lithium chloride (399). Modifications include the use of paper chromatography (636) and fluorescence measurements a t 518 mp before and after photolytic reduction in the absence of air (356). Other papers deal with the absorption and fluorescence spectra of riboflavin in solid sodium silicate solution (182), of riboflavin, riboflavin tetrabutyrate, and lumichrome in organic solvents (370), and the photochemical degradation of riboflavin and related compounds (726). Details have been given for determining pyridoxal and its 5-phosphate in blood or body organs by the fluorescence resulting from reaction with cyanide in phosphate buffer (724). The effects of p H on the excitation and emission peaks of pyridoxol, pyridoxal and its 5-phosphate, pyridosamine, pyridoyic acid and its lactone, as well as several related compounds, have been described (96, 452). Chen found that the fluorescence efficiencies of several of these vitamin B6 compounds are highly dependent on temperature, and the compounds are very sensitive to light (118). Thiamine fluorometry via thiochrome has been the subject of many papers, covering the analysis of foods (455, 546, 669),automated procedures (340), and paper chromatography separation of thiamine from its phosphate and pyrophosphate (91, 251, 4 2 ) . The customary method is to convert thiamine into thiochrome by alkaline ferricyanide oxidation, which produces fluorescence from hydroxyalkylthiamines as well as thiamine itself, but only the latter will give fluorescence if cyanogen bromide is used in place of the ferricyanide (608). The amount of thiochrome formed by atmospheric oxidation in old thiamine solutions has been taken as an indication of the degree of spoilage (56Q). For the assay of thiamine disulfide, cysteine serves to reduce the disulfide bond to sulfhydryls before the thiochrome is developed (429); in thiolized thiamine, a stable red-fluorescent product suitable for quantitation is produced with greater sensitivity if hydroxylamine is added as a reducing agent to the cupric ion solution (728). Catecholamines. Much interest is still being shown in t h e differential determination of adrenaline, noradrenaline, and related compounds. T h e basic principle involves oxidation t o the fluorescent trihydroxyindoles, with stabilization of t h e intensity by a mild reductant. After alkaline ferri-

cyanide oxidation, both adrenaline and noradrenaline will give fluorescence if ascorbate is the stabilizer used, but only noradrenaline will be measured with thioglycollate stabilizer (552, 685). Other approaches to the differential estimation use variations of p H and fluorometric wavelengths, or iodine as the oxidant (328, 427, 428, 513). Sodium borohydride has been proposed as an additional stabilizer, and the importance of temperature control in the trihydroxyindole method has been noted (236). Automated procedures for plasma catecholamines have been described (213, 214) as well as a rapid screening test for urine (206). Inasmuch as catecholamines may occur in conjugated as well as free form in urine, hydrolysis is required if the total is to be reported (226).

-4nton and Sayre have worked out a procedure for the catecholamine metabolites metanephrine and normetanephrine, which are the 3-methoxy derivatives (18); both are oxidized to fluorescent products by periodate at p H 5, but only the nietanephrine gives fluorescence when oxidized a t pH 1.5. Another trihydroxyindole procedure for these compounds in urine is given by Kahane (326). Some in vivo oxidation products of adrenaline and noradrenaline have been detected (358). Cnder appropriate conditions, catecholamines and other substituted phenylethylamines when condensed with formaldehyde yield green fluorescent products of potential use in histochemistry (139) and assay on paper (47). Other Amines. I n the ethylenediamine fluorometric method for dopamine (the acronym for 3,4-dihydroxyphenylethylamine, also known as 3-hydroxytyramine) modifications t o improve the specificity have been described (391). Dopamine and 6-hydrosydopamine have been determined in the presence of each other by the difference in emission spectra of their ethylenediamine condensates (392). As noted just above, m-hydroxyphenylethylamines like dopamine or m-hydroxyamphetamine can react with formaldehyde to give fluorescent 3,4dihydroisoquinolines (139, 321). Tyramine and related compounds have been determined fluorometrically in stored post-mortem livers (324). I n the urine of schizophrenic patients, 3,4-dimethoxyphenylethylamine was determined by its fluorescence a t 640 nip in alcoholic KOH, with escitation at 290 mp (686). Because of the physiological importance of serotonin (5-hydroxytryptamine), many reports on its fluorometric determination continue to appear. These include condensation with ninhydrin (432) or o-phthalaldehyde (412, 738),extraction with acidic butanol and

measurement of the intrinsic fluorescence (716, 717), reducing the lightscattering error by the use of crossed polarizers (3), and an improved tissue blank by reading the intrinsic fluorescence in 3N HCl, before and after adding ferricyanide which destroys only the serotonin but no other substance fluorescing a t 545 mp under 295 mp excitation (14). Glick, Von Redlich, and Diamant describe an adaptation suitable for nanogram amounts of serotonin (244). Tryptamine itself can be determined fluorimetrically in urine with good linearity (507). An automated method for histamine in blood (562), a simplified ion exchange technique (306), and a fluorescence microscopy method for histamine in tissues (322) are based on the fluorophore formed by condensation with o-phthalaldehyde. X detailed study of this reaction was made for the purpose of finding optimum conditions for its use in a chromatographic spray reagent (601). A similar method was employed to follow the release of histamine from leukocytes (531). Medina and Shore have improved the specificity of the original o-phthalaldehyde procedure by first removing spermidine (440). The same reagent can be used for fluorometry of spermidine itself (198) and simultaneously with histamine, by prior separation on a phosphorylated cellulose column and elution with different dilutions of HC1 (580). A wide variety of primary and secondary amines can be detected a t very low concentrations by the fluorescence of their 1-dimethylaminonaphthalene5-sulfonic acid derivatives, after separations by thin layer Chromatography (593). For the fluorometric determination of primary aromatic amines, 2,2naphthalenedialdehyde is suggested as a condensing reagent instead of 0phthalaldehyde (10). A procedure for microgram quantities of ethylenediamine residues in milk is based on the reaction with adrenochrome to yield a fluorescent compound, with the emission peak a t 510 mp under excitation a t 460 mp (496). It appears possible to determine such biogenic amines as spermine, putrescine, or cadaverine by the reaction with resorcinol and acrolein, followed by fluorometry of the product ( 5 ) . Krasovitskii has studied the absorption and fluorescent spectra of the azomethine derivatives of certain aromatic amines (375,376) and the effect of temperature on such spectra of 4aminophthalimide has been reported (330). Heterocyclic imines and polynuclear aromatic amines can usually be detected on chromatographic plates by the color change or intensification of the fluorescent spots when moistened with tetraethylammonium hydroxide (575).

Amino Acids and Proteins. A microfluorometric determination of arginine, based on its reaction with ninhydrin in alkaline solution, has been described as useful in histochemistry (557). Reduced glutathione in blood was assayed with good specificity and sensitivity by condensing with o-phthalaldehyde a t p H 8 and measuring the fluorescence produced (135). Enzymic procedures have been detailed for the determination of glutamate, aspartate, and a-aminobutyrate in the mole range by measuring the fluorescence of the NADH or related nucleotide formed in the coupled enzyme reaction (265, 256). The intrinsic fluorescence of 4-sulfoanthranilic acid a t 440 mp was employed for its determination, after elution from the paper chromatogram (483). A bacterial cell wall constituent, a,e-diaminopimelic acid, can be determined in nanogram amounts by fluorometry of the o-phthalaldehyde reaction product (554)*

Location and quantitation of amino acid spots on thin layer chromatograms is facilitated if the compounds have been made highly fluorescent by labeling with the dimethylaminonaphthalenesulfonyl group (173, 454, 504). Studies on such compounds include the effects of pH on the fluorescence spectra (385), the role of solvent dielectric constant (120), and the quantum yield (121). By fluorometric titration, it was found that five moles of 1-anilino-&naphthalenesulfonate are bound to bovine serum albumin, and the effects of temperature, pH, and ionic strength were noted (169). The clinical importance of avoiding mental retardation by early detection of phenylketonuria in infants continues to generate papers on the fluorometric determination of phenylalanine (11). Among the details considered are: deproteinization of the blood by steaming (75, 310), automated procedures involving elution of the dried blood Samples from filter paper collection cards (131, 286, 287), and an automated method based on the fluorescence produced by reaction with ninhydrin (612). Tyrosine blood levels are often required clinically as an adjunct to the phenylalanine levels; an automated fluorometric procedure for tyrosine is based on a modification of the Waalkes and Udenfriend method of coupling with 1-nitro-2-naphthol (292). Tryptophan has been determined in plasma and urine by its fluorescence after thin layer chromatography (239); it can also be assayed with high sensitivity by conversion to the fluorophore norXanthurenic acid, harman (169). which is a urinary metabolite of tryptophan, has been determined by its intrinsic fluorescence in a shortened method which substitutes iso-butanol extractions for chromatographic sep-

arations (134). Several studies have been made on the effects of temperature, pH, and ring substituents on the fluorescence, phosphorescence, and quenching of tryptophan and other aromatic amino acids (58, 124, 615, 661). Structural changes in proteins cause changes in the fluorescence of their tyrosine and tryptophane constituents; Cowgill has found that the denaturation of RNase (142) and the reductive cleavage of disulfide links (141) give an increased intensity of such fluorescence. However, several proteins in 96'% ethanol showed a variation of fluorescence with temperature (20" to -183°C) which differed from that of tryptophan (243). Helix-coil transitions in polypeptides and a vinyl polymer have been followed by measuring the polarization of the fluorescence emitted a t various p H values and temperatures (240, 241, 688). Photochemical changes in aromatic amino acids and proteins were studied by comparing the initial and final fluorescence spectra a t 77" K (684), with the finding, for example, that kynurenine is probably formed by photolysis of tryptophan. In a study of protein conformation with 2-p-toluidinylnaphthalene-6-sulfoliate as a fluorescent probe, the emission intensity was seen to increase dramatically when chymotrypsinogen was activated to chymotrypsin, by means of trypsin (437); fluorometric kinetics of activation followed the Michaelis-Menten relation. Low temperature luminescences of collagen have been contrasted with those of RNase and human serum albumin (293). Fluorescence decay times of several proteins and other biochemical molecules have been measured in the nanosecond range (116). Serum proteins in chromatographic column effluents can be collected automatically by a device which uses their native fluorescence (658); they can also be quantitated in electrophoresis patterns by fluorescent staining (599).

Enzymes, Nucleotides, and Nucleic Acids. Guilbault has reviewed the uses of enzymes in analytical chemistry, with some emphasis on fluorescence techniques (261). With coworkers, he has also published analytical procedures for peroxidase by oxidation of homovanillic acid to a fluorescent dimer (262), for hyaluronidase by conversion of indoxyl acetate into strongly fluorescent indigo white (263) and for lipolytic enzymes by the liberation of fluorescein or eosin from their nonfluorescent esters (264). For hydrolytic enzymes, widespread use is still being made of the just mentioned technique of generating and measuring a fluorescent product from a nonfluorescent substrate. Thus, p-glucuronidase has been determined by fluorometry of the 2-naphthol (257) or VOL. 40, NO. 5 , APRIL 1968

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4-methyl-umbelliferone (720) liberated from their glucuronides; leucocyte alkaline phosphatase, similarly with 2-naphthyl hydrogen phosphate as the substrate (438); p-xylosidase, by 4methylumbelliferyl p-D-xyloside (167) ; and lipases, by fluorescein dibutyrate (568) or 4-methylumbelliferyl fatty esters (314) as substrates. Other examples of the same type are the fluorometric determinations of dipeptidyl arylamidase (439), unspecific esterases (126), and N-acetyl-p-glucosaminidase (676, 719).

For assay of dehydrogenases and similar enzymes, another general technique is very useful. As indicated earlier, the reactions of these enzymes involve oxidation or reduction of the nicotinamide adenine dinucleotides (XAD, X-kDH, N.kDP, and KADPH) which result in sensitive changes in the fluorescence of thebe compounds, measured under specified conditions. Among such recent procedures for fluorometric determination of enzymes are those for serum lactate dehydrogenase (498, 686) and its isozymes (50), alcohol dehydrogenase (463), malic dehydrogenase (586), a-hydroxybutyrate dehydrogenase as a confirmation of myocardial necroses (48),glutamicpyruvic transaminase (280), succinate semialdehyde dehydrogenase (515), and galactose-lphosphate uridyl transferase, absence of which causes galactosemia (54). Experimental details have been given for the determination of eleven enzymes (408) and sixteen cofactors or substrates (409) involved in glycolysis in the brain. Thiaminase activity in fish was measured by fluorometry of the undestroyed thiamine a t the end of ten minutes (246). The assay of serotonin sulfokinase was based on the fluorescence of the serotonin sulfate formed (286). Other fluorometric procedures have been published for the determination of monoamine oxidase (372), tyrosine hydroxylase ( I ) , and creatine phosphokinase (136, 603). The substantial change in fluorescence intensity that follows the reaction of cycloserine with pyridoxal-5-phosphate was used to monitor the release of the cofactor from the active site of the enzyme aspartate-glutamate transaminase (129). Staining of bacterial cells by fluorescein-labeled @-glucosidaseappeared to depend on the accessibility of glycosidic linkages in the cell wall (516). Fluorescence was used to determine the asqociation constants of lysozyme with several saccharides containing N-acetylglucosamine and N-acetylmuramic acid (125).

Pyridine nucleotides and related compounds have been determined fluorometrically (543, 741). Chen and Hayes have discussed methods of handling high concentrations of N-kDH and NADPH in the spectrofluorometer 126 R

ANALYTICAL CHEMISTRY

(119). An assay for organic phosphorus

was based on the stoichiometric generation on TU'ADPH (348). Procedures were given for fluorometric determination of adenosine diphosphate and triphosphate with the firefly luciferase system (181, 295) and also for the flavine nucleotides (423). N a n y studies have been reported on the luminescence characteristics of the purines, pyrimidines, nucleosides, and nucleotides (27, 64, 65,132,133, 278,353, 407).

The phosphorescence of DSA (deoxyribonucleic acid) from different sources, measured at 77" K in 0.03M sodium chloride in 50% glycerol-water, increases with the number of adeninethymine base pairs, and is quenched by many divalent cations (311 ) . Soluble RX-1 from E. coli shows about 100 times less phosphorescence than either polyadenylic acid, or the same s-RN.4 after hydrolysis, possibly because of triplet energy transfer from purines to pyrimidines in thc hydrogen bonded base pairs (629). Acridine Orange forms complexes with DNA and RNA, for which the fluorescence and excitation spectra have been recorded; for a 100: 1 ratio of dye to nuclei acid, the Acridine Orange dimer originally present in solution dissociates into monomer, which gets intercalated between the base pairs, giving enhanced fluorescence (547, 657). The Acridine Orange complexes with native and denatured DSA have emission peaks at 530 and 640 mp, respectively, which makes it possible to determine as little as 2% denaturation in a native DNA sample (71). A new fluorometric method for R S h and DSA determination, based on the great increase in fluorescence of ethidium bromide (2,7-diarnino-9-phenylphenanthridine-10-ethyl bromide) upon binding to nucleic acid, was described by LePecq and colleagues (401). They have also used ethidium bromide for following the synthesis of DNA-RKh hybrids in vitro, because the binding fluorescence depends on the secondary structure of the nucleic acids (400). Another quantitative determination of DNA, in cell nuclei, utilized the greenish yellow fluorescence of the complex with auramine 00 dye (661). Steroids and Hormones. The general procedure for fluorometric determination of corticosteroid hormones is based on ethanol-H2S04 treatment to develop the fluorescence. Refinements of this and related methods have been described for cortisol and corticosterone in various body fluids and tissues (179, 180, 193, 223, 242, 257, 430, 486, 689, 581, 693, 752). A similar procedure was used for aldosterone (235). Ittrich has proposed a new method for deoxycorticosterone, in which the fluorescence is developed by 2% hydroquia t 100' C and exnone in 68%

tracted by benzyl alcohol, as being linear from 10 to 200 micrograms and showing little interference from cortisone, progesterone, and some other steroids (312). Corticosterone and testosterone were observed to form a fluorescent complex with nicotinamide adenine dinucleotide, but androsterone and tetrahydrocortisol did not (210). Improvements continue to be reported in the fluorometric determination of estrogens, such as estradiol and estriol, by treatment with sulfuric acid (463, 588, 613, 634). Urinary estrogens can also be assayed with hydroquinone in H2SOa, followed by nitrophenol in chloroform, and measurement of the fluorescence at 578 mp under 546 mp excitation (269). The natural fluorescence polarization of estradiol, estriol, and other steroids has been studied (635). Optimum time and temperature conditions for inducing fluorescence in testosterone with sulfuric acid have been determined, and the fluorescence and excitation spectra reported for testosterone, aldosterone, cortisol, and corticosterone (234). Fluorometric analyses of cholesterol in blood made use of the fluorescence developed in chloroform solution by treatment with acetyl choride-zinc chloride reagent (654) or acetic anhydride followed by sulfuric acid (740); an automated procedure has also been described (355). Sterol acetate spots on thin layer chromatograms fluoresce brightly after spraying with 0.2% ethanolic dibromofluorescein (138). Cholic, deoxycholic, and other bile acids have been determined by their fluorescence after treatment with HzS04 of various concentrations (220,478,626). An assay of ACTH was obtained by incubation with bovine adrenal cortex in vitro and fluorometry of the released cortisol by the ethanol-H2S04 method (582). The purification of bovine growth hormone can be followed by means of the six-fold increase of its fluorescence (because of the tryptophan residues) upon acidification from p H 5 to 2.5 (106). Among the plant hormones, gibberellic acid in rhubarb was determined with a sensitivity of 3 ppb by its fluorescence on thin layer chromatograms (3.49, and indoleacetic acid and many related indoles were determined at 10-6.U or lower concentrations, through use of their intrinsic fluorescence (104, 534).

Pharmaceuticals. The ergot alkaloids (368, 565) and cinchona alkaloids (660) have been assayed, on thin layer chromatograms for example, by means of their fluorescence. Alkaloids having a primary or secondary amino group or a phenol group will react with 1dimethylamino-naphthalene- 5 -sulfonyl chloride t o produce a green fluorescence in alkaline solution, which can be

extracted with ether or chloroform and quantitated (691). Quinine, reserpine, and hydrastine have been determined by a combination of thin layer chromatography and fluorometry (533). Fui ther experience and possible interferences in the fluorometric microdetermination of streptomycin by reaction n ith sodium p-naphthoquinone sulfonate have been discussed by Faure aiid Blanquet (209). I n study of the fate of tetracyclines in biological systems, their intense fluorescence in the piesence of calcium or magnesium ion was found to be most valuable (534). An automated fluorescence assay a t 420 mp (excitation a t 350 mp) has been described for Antimycin A, ai1 antibiotic which kills teleosts (592); griseofulvin also is determined by its intrinsic fluoresceiic 3 (215, 3?3). Fluorometric procedures have been reported for amphetamine, by condens(569, ing with formaldehyde in aiid for pentobarbital, in 0.5Y NaOH solution (590). Barbiturates in tablets can be detected by the greenish fluorescence they give 17 hen heated with resorcinol and the characteristic changes caused by adding strong acid or base (602). Submicrogram amounts of lysergic acid diethylamide have been determined fluoronietrically, even in the preqence of other drugs (156, 233, 502). Holmstedt has shoir n that epena, a South .knericari intoxicating snuff, contains A',S-dimethyltryptamine and its S-hydroly and 5-methoxy derivatives, and has given the fluorescence spectra for all three (296). Phosphorimetric determination for cocaine, procaiiie, phenobarbital and chlorpromazine in blood serum have been described (714). Chlorpromazine and other phenothiazine drugs in urine were analyzed by spectrofluorometry, based on the four-peak excitation spectrum aiid single-peak emission after treatment with permanganate (444). Certain S-(w-dimethy1amiiio)alkyl derivatives of phenothiazine, when treated n i t h H2S04 and then diluted with dimethyl sulfoxide, develop a fluorescence which permits their detection in submicrogram quantities (420). Fluorometric and phosphorimetric emission and excitation peaks and decay times are given by Hollifield and Winefordner for warfarin, dicumarol, diphenandione, phenindione, and tromexaii (294); excellent recoveries from whole blood were achieved for dicumarol by phosphorimetry and for warfarin by fluorometry. I n a plasma assay for warfarin, which is 3-(a-acetonylbenzyl)4-hydroxycoumariiiJ the fluorescence waq enhanced by acetone as solvent instead of water (140). Fluorometric procedures have also been published for indoxle, an oral anti-inflammatory agent (327); 9aminoacridine, in ethanolic HC1 (560);

ethacridine lactate in 0.01N HzS04, with the emission peak at 515 mp (271); imipramine and other dibenzazepine derivatives (168, 419) ; chloroquine and other 4-aminoquinolines either directly or after oxidation with ferricyanide (201, 222); guanisoquin, by coupling with ninhydrin in alkaline solution (111); and 2-PAM, a nerve gas antidote, after alkaline hydrolysis to 1methyl-2-pyridoneJ which has excitation and emission peaks at 315 and 370 mp, respectively (238). Sulfanilamide was determined with good sensitivity and specificity by the fluorescelice of its condensate with 4,5-niethylenedioxyphthalaldehyde(9). The Peters method for isoniazid in serum has been simplified and adapted to a simple fluorometer (544).

Agricultural Chemicals and Products. Boivman and Beroza have

tended the determination of aflatoxins down into the subnanogram range (26, 510, 528). Andrellos, Beckwith, and Eppley have found that ultraviolet irradiation causes photolysis of aflatoxins B1 and GI, which proceeds faster on the silica gel than in methanol solution and yields products of lower Rf value and toxicity (17). The relative fluorescence intensities of aflatoxins B1, Bz,GI, and Gzare different in the three solvents methanol, 95% ethanol, and chloroform (651). A spectrofluorometric determination of o-phenylphenol in the rind of citrus fruits has been reported (535). wolf and Stevens described the fluorescence characteristics of p-carotene, lutein, lutein epoxide, and violaxanthin; these carotenoids were extracted from wheat seedlings and separated by paper chromatography (718 ) .

presented fluorescence and phosphorescence spectra for methylenedioxyphenyl synergists used in insecticides, and have devised methods for piperonyl butoxide and sulfoxide in various products (78). They have also published the spectra of 17 insecticidal carbamates, and described a rapid fluorometric determination for five such carbamates in milk (79). Phosphorescence spectra, decay times, detection limits, and analytical curves were presented for 32 pesticides and their degradation products; 20 others tested showed no phosphorescence (456). Guthion (azinphosmethyl) in milk aiid animal tissue was assayed by alkaline isopropanol hydrolysis and fluorometry of the anthranilic acid formed ( 2 ) . Naretin (N-hydroxynaphthalimide diethyl phosphate) in meat or fat was similarly hydrolyzed by 0.5AV XaOH in methanol and then quantitated by the fluorescence produced (15). Of 47 orgaiiothiophosphorus pesticides tested, 32 gave blue fluorescent spots on thin layer chromatograms after exposure to bromine vapor and spraying with ferric chloride and 2-(o-hydroxyphenyl)-bencoxazole (532). Chlorinated insecticide spots likewise can be visualized by fluorescence after the chromatogram is sprayed with such reagents as N-methylcarbazole or Rhodamine B (42). Buquinolate (ethyl 4-hydroxy-6,7-diisobutoxy-3-quinoline carboxylate) in chicken feeds was determined by a chromatography-fluorometry method, with 99.7y, recovery (70). Aflatoxins are highly toxic and fluorescent materials formed by A . jiavus mold growth on peanuts and other agricultural products; the usual analysis employs fluorometry at 425440 mp after silica gel thin layer chromatography with chloroform containing a few per cent of methanol as the solvent system (511, 624, 659). Objective photoelectric measurement of the fluorescence of the spots has ex-

Immunofluorescenceand Biological. A versatile technique for detecting or quantitatively determining microorganisms or many molecular species is to use them as antigens for stimulating specific antibody production (in rabbits for example), then fluorescence-label the antisera by reaction with fluorescein isothiocyanate, then stain the preparation under study with the labeled antibodies and examine by fluorescence microscopy to note the amount and location of the antigen-antibody reaction. Such immunofluorescent techniques, with various refinements, have been used for influenza virus (467), oncogenic viruses (461), streptococci (291) including those which cause dental caries (315), an ecological study of bacilli in soil (289),dysentery bacteria in water (656), and diagnosing histoplasmosis (300) and trichinosis and schistosomiasis (639). Other applications reported include blood group differentiation (462), detection of two types of hemoglobin (A and F) in a single erythrocyte (159), assay of penicillin antibodies (160, 161, 529), and the localization of transferrin in liver (587), of amylase, trypsinogen, and chymotrypsinogen in the acinar cells of pig pancreas (727),and of nucleosides such as adenosine and guanosine in the nuclei of mouse L cells (352). A principal difficulty in immuaofluorescence is nonspecific staining, which can be minimized by careful purification and other techniques (100, 277, 466). Fothergill has reviewed the properties of fluorescent protein coiijugates and discussed the excitation and emission spectra and p H effects, as well as the immunological aspects (221). The purity of fluorescein isothiocyanate and its quantitative fluorescence intensity in the free and protein-bound states have been investigated (645,668). I n the biological field, fluorescence techniques were used to study oxidative and glycolytic recovery metabolism in VOL. 40, NO. 5 , APRIL 1968

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muscle (32O), the movement of water in willow and related plants, with Pontacyl Brilliant Pink as the tracer dye (553), human arterial elastin constituents (61), the relative permeability of mammalian cell membrane to fluorescein and fluorescein diacetate by esteratic activity within the cell (568), nucleic acid quenching of carcinogenic hydrocarbons (609),and possible automated cytology for cancer screening by fluorometry of unbound acriflavine used as a nucleic acid stain (560). Of the large number of publications on the fluorescence of chlorophyll and its relation to photosynthesis, only a few are listed here (97, 98, 247, 460). The phosphorimetric background of ether extracts of blood and urine a t various pH values has been explored in detail (435), and fluorescent substances in normal urine have been identified (216). Miscellaneous. The relationship between fluorescence yield and molecular structure was studied for a group of 20 rhodamine dyes (683). Rau described the very intense fluorescence of certain azo compounds in concentrated acids (536); Steel and Thomas measured the spectra and lifetime of the first ewited singlets of two cyclic azo compounds (627). Solvent effects on the fluorescence of 3-amino-N-methylphthalimide (677) and 4-amino-N-methylphthalimide (681) have been studied. I n thin layer chromatography of brominated salicylamides, fluorescence was used in examining the spots (618). Organoleptic and fluorescence analyses have been compared for the impurities in fermentation alcohol (38). The characterization of olive oil refining processes by spectrofluorometry of the oil has been discussed critically (269, 323). Improvements have been made by Kurchatkina in the use of fluorescent indicators in the determination of aromatic and olefinic components of petroleum distillates (381, 382, 383). Distinguishing oil-bearing from waterbearing rock formations was assisted by evaluation of the luminescence characteristics (424). The acidity distribution on catalysts was visualized with the help of fluorescent indicators (187], and flotation agents on the surface of coal particles were determined fluorometrically with high sensitivity (632). The infrared fluorescence of coals and graphite has been determined photographically (224). A simple fluorometric determination of lignin sulfonates in spent sulfite liquors from paper-making shows a linear concentration dependence down to 0.2 ppm (128). A sensitive and specific assay of bilirubin in serum measures the fluorescence at 500 mp under excitation a t 435 mp after treatment with 85% H3PO4(559). The absorption and emission of phthalocyanines have been recorded (192). 128 R

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Strzelecki has reviewed the subject of polymer fluorescence (638). Related publications deal with structural effects (229), attempted determination of molecular weight in linear polyesters (231), the increase in acetylated cellulose luminescence with degree of acetylation (230), and fluorescence polarization in the study of segmental motions in concentrated polymer solutions (647). The fluorometric determination of malvin content in wines and grape juice, which aids in characterization, can use either its own fluorescence (196) or that induced by nitrous acid, followed by ammoniacal ethanol (56, 268). Nitzsche has briefly reviewed the occurrence of annuloline, the alkaloid responsible for fluorescence in perennial ryegrass (47 1 ) . I n gobelins (historical textiles), repaired and restored areas are recognizable by fluorescence analysis (397). Winkelman and Grossman have devised a front face optical configuration for the quantitative fluorescence analysis of tetraphenylporphinesulfonate, tetracycline, etc. in turbid or opaque buspensions (715). Improved methods have been proposed for fluorometry of chromatograms (74, 307). Low temperature fluorescence and phosphorescence data for 59 organic compounds were reported (127). Chemiluminescence phenomena continue to attract the attention of many workers (110, 411, 637). Subnano,v a m quantities of hemoglobin were determined by the catalytic effect on the chemiluminescence of luminol ( 4 6 3 , and the inhibitory effect wa3 employed to determine amino acids (626) and aromatic nitro-, amino-, or hydrosycompounds in some binary mixtures (527). Also used for analytical purposes were the cheniiluminescence produced by oxidation of gallic acid or pyrogallol by ferricyanide and hydrogen peroxide (620),and the effect of formaldehyde concentration on the orangechemiluminescent reaction with peroxide and gallic acid (621). Glowacki has discussed the mechanisms of molecular interactions in fluorescence quenching processes (245). Radioluminescence has served for the determination of styrene, benzoic acid, bromoform, and other compounds, using plastic and liquid scintillators (559, 360).

LITERATURE CITED

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