Fluorometric Analysis Alfred Weissler F o o d a n d Drug Administration, Washington, D.C. 20204
This review is dedicated to t h e memory of Prof. Charles
E. White, who was at first the sole author and then the
senior author of the 13 previous biennial reviews in this series. He was a leader in establishing the value and importance of fluorescence analysis; he was also a good and warm-hearted person, whose death in May 1973 was a real loss. This article covers primarily the literature indexed by Chemical Abstracts during the two years from December 1971 to December 1973. Just as was true in the last biennial review (985), many hundreds of publications on fluorescence have had to be omitted; limitations of space permit mention of only those papers that may be useful for fluorometric analysis. Atomic fluorescence and X-ray fluorescence are covered elsewhere and are not included here. An interesting recent development is the use of vacuum ultraviolet radiation; such high-energy excitation promises to extend the applicability of luminescence analysis (979). Other reviews have been published on theoretical aspects of absorption and luminescence spectra (685, 808), special kinds of fluorescence such as delayed and sensitized (973), nanosecond fluorescence spectroscopy of macromolecules ( I O I O ) , and an index of radiative lifetimes of many fluorescent ions, molecules, and radicals (786). Guilbault has authored several comprehensive treatises on fluorescence spectrometry (303, 304, 306), and Winefordner has published a review of phosphorimetry in a periodical which contains several other good papers presented in March 1972 at a National Bureau of Standards symposium (998). The world-wide interest in fluorescence analysis is attested by reviews in many foreign languages: in Finnish (873), in French, on biochemical applications (186), in Greek (478), in Japanese (1006), in Russian (211, 703), and in Serbo-Croatian, on pharmaceuticals (896).Fluorescence spectroscopy has been reviewed in terms of its fundamentals (132, 133), its use in studying protein structure (720) and drug interactions with biological systems (135), and as a technique for drug analysis (515). Van Duuren has written a review on phosphorescence spectroscopy and its uses in biochemical analyses (941). Low-temperature luminescence analysis for inorganic trace impurities has been reviewed (840),as have also the spectra and energy transfer of rare earths in inorganic glasses (737) and the photochemistry and luminescence of coordination compounds (611). Bartos and Pesez have given 169 references in their review of organic analysis, with special attention to functional group fluorometry (56); p H effects in luminescence spectroscopy have been discussed by Schulman (789). Other reviews deal with quasilinear luminescence and absorption spectra of polyatomic organic molecules (764), fluorescence and phosphorescence spectra of organic crystals (281), concentration depolarization of fluorescence (441), detection of ultraviolet (UV) absorbing substances with fluorescent materials (889), and the usefulness of fluorometry in the analysis of foods (61, 116), antibiotics (706),pharmaceutical preparations (447), and in the clinical chemistry laboratory (757). Konev’s book on “Fluorescence and Phosphorescence of Proteins and Nucleic Acids” has been translated from Russian into English (469). Related reviews discuss fluorescence studies of biopolymer structure (94, 119, 768), the luminescence properties of polynucleotides (462) and DNA (deoxyribonucleic acid) solutions (916),the cytophotometric determination of DNA by fluorescence (79), and fluorescence studies of the conformation of nucleic acids (120). Of the many publications each year on the relation between chlorophyll fluorescence and photosynthesis, three reviews are noted here (190, 273, 288). Excited states and 500R
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luminescences of chlorophylls, carotenoids, and other photobiological molecules have been compared as to theory and experiment (846). “Chemiluminescence and Bioluminescence” is the subject of a 1973 book edited by Cormier, Hercules, and Lee (143),as well as of a review in Japanese (285). Ultraweak bioluminescence continues to attract Russian workers, for example in relation to lipoproteins in biological membranes (890, 1020).Weber has reviewed the uses of fluorescence methods in biophysics (972), and Zaharia has discussed spectrofluorometry in biology, in Romanian (1015).
INSTRUMENTATION AND TECHNIQUES Melhuish has discussed the problems of putting spectrofluorometry on an absolute basis (600). Various types of standard materials for luminescence measurement have been described, including organic compounds in solution (951), inorganic ions in glasses (738), and thallium-doped potassium chloride disks (740). Consideration has been given to several aspects of fluorometric standardization in clinical chemistry (677). Representative papers on the effects of various experimental parameters in fluorometry deal with temperature (572), concentration (944), excitation wavelength (52), bandwidth of exciting and emitted light (957), scattered light (668), the solvent matrix in phosphorimetry (550), and fluorescent impurities leached out of polyethylene containers of aqueous reagents (131). Several new sample cells have been designed, including a continuous flow cell for fluorometry (869), a capillary cell for phosphorimetry in aqueous solvents a t 77 OK (551), and windowless front-surface cells for fluorometry (588) and low temperature luminescence measurements (589) of turbid or highly-colored solutions or of pastes. Fluorescence instrumentation in general has been discussed (301), as well as improvements for phosphorimetry (549).Many new instruments have been designed, such as those for multiple internal reflection fluorometry (338), diffuse reflectance and fluorescence measurements on 20-mg samples of powder (283), high resolution simultaneous recording of total luminescence and phosphorescence spectra (959), quantitative microspectrofluorometry (310), wavelength programming (651),and measurements of luminescence polarization (510) and circular polarization (855). A high sensitivity filter fluorometer of small size was developed, using semiconductor components and integrated circuits (202). Computerization has been adopted widely, for such purposes as simultaneous absorbance and fluorescence measurements (366), digital integration fluorometry of photochemically unstable compounds (548), and automatic digitization and correction of fluorescence spectra (780, 824). Argon laser excitation has been employed in a low-noise luminescence spectrometer (989) and for comparing the absolute quantum yields of Rhodamine 6G in various solvents (16). A COz laser was used to excite the infrared fluorescence of ethylene, and the use of this technique in monitoring air pollutants was proposed (747). The value of fluorescence or phosphorescence lifetimes for additional characterization of the emitting species has stimulated much work on the measurement of lifetimes. Pulsed source, time resolved phosphorimetry succeeded in distinguishing structurally similar aryl ketones with nearly identical emission spectra (228, 331). Electronic circuitry for converting spectrofluorometers into time-resolving phosphorimeters (327) and for computerized study of phosphorescence decay (413) were described. Measurement of radiative lifetimes, mainly in the nanosecond range, have utilized fast-scan spectrometry ( I l l ) , a modi-
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Alfred Welssler is director of the Division of Color Technology at the Food and Drug Administration. He received a PhD in 1946 from the University of Maryland in analytical-inorganic chemistry, with a minor in physics, where his thesis work on the fluorometric determination of aluminum and boron was directed by Charles E. White. Earlier degrees were an MS in organic chemistry from the University of Wisconsin in 1938 and a BS in chemistry and biology from the City College of New York in 1936. On a part-time basis he was assistant professor of chemistry at the University of Maryland for several years and is now chairman of the Physical Sciences Departmental Committee of the U.S. Department of Agriculture Graduate School. His career has also included posts at the Naval Research Laboratory, Army Research Office, National Institutes of Health, and Air Force Office of Scientific Research. In addition to his work in fluorescence analysis, he has studied the applications of ultrasound waves in chemistry.
fied phase-shift technique (405),single photon methods (64, 793),and pulse-sampling oscilloscopes (577,660) in some cases with instruments suitable for use over an extended range of temperatures (302,900).A high-intensity 10-nsec light source was made by discharging a coaxial capacitor in air (1028).Laser excitation made possible some fluorescence kinetics studies in the picosecond range, on chlorophyll (802)and various dyes (37). In high-speed liquid chromatography, several workers have achieved a good increase in sensitivity by use of a fluorescence detector in place of, or in addition to, the usual UV absorption detector (126, 342, 676).Even if the compounds in the effluent are not intrinsically fluorescent, it is still possible to use a fluorescence detector if (a) the compounds are oxidizable by Ce(1V) with the production of Ce(II1) which is fluorescent (436,437), or (b) the compounds can be labeled with a fluorophore reagent such as 5-dimethylaminonaphthalene-1-sulfonyl (DANSYL) chloride (240).Similarly, in gas chromatography, fluorescent detectors for the effluent vapors provide higher sensitivity and specificity, as for polynuclear arenes, than other detectors (108, 238, 748). Refinements continue to be made in fluorometric quantitation of thin-layer chromatograms (TLC) (353);as a n example, a tenfold greater sensitivity for the detection of pyrene was found on alumina TLC plates than on silica gel plates, which shows the importance of the adsorbent as well as of other experimental parameters (682).The effect of humidity and other variables in TLC on the fluorescence intensity of DANSYL derivatives was studied (88). It was found possible to obtain strong triplet-state phosphorescence a t room temperature, avoiding the usual need for low temperatures, by using a n enhancer of spin-forbidden transitions (dimethylmercury) as a solvent, in the case of polynuclear aromatics (945),or by using adsorption on paper, silica, alumina, etc. followed by thorough drying, in the case of acid or amine derivatives of polynuclear compounds (787). Mathematical treatments of the combined luminescence spectra in the analysis of multicomponent systems have been proposed (698, 826). Other papers on techniques deal with a new source of intense broad-band ultraviolet, produced by directing a n electron beam into superfluid liquid helium (230);the use of tetraphenylbutadiene instead of sodium salicylate as a wavelength converter for the vacuum ultraviolet (115); the photochemical generation of stable fluorescent compounds, as by photoelimination of CO and C02 from anthracenedicarboxylic anhydrides (1026);experimental methods for luminescence measurements on aromatics in rigid plastic media (997);observation of X-ray- and UV-induced emission during irradiation as well as afterwards, over a range of temperatures (852); quantitative analysis of samples by measuring bioluminescence or chemiluminescence (571); counting bacteria in urine samples by bioluminescence measurement of bacterial adenosine triphosphate (ATP) (445);and sorting bacterial cells after rendering them differentially fluorescent (85).
INORGANIC An interesting development in recent years is the extension of analytical fluorometry into gas-phase and solidphase determinations, to supplement the traditional liquid solution techniques. Solid-phase methods have been applied particularly to the analysis of rare earth impurities in crystals and glasses, sometimes with X-ray excitation of the optical luminescence (163);further details are given in later sections. Fluorometric analysis in the gas phase has been stimulated by the growth of upper atmosphere and space chemistry. The far ultraviolet fluorescence of sulfur dioxide (93)was made the basis of an SO2 detector which uses excitation by the Zn 215-nm line or the Cd 229-nm line, the latter giving a linear relation between fluorescence intensity and concentration over the range 0.1 to 1600 ppm (655).Measuremdht of the SO2 intensity as a function of excitation wavelength over the range 200-230 nm showed a sudden decrease below 219 nm, where the higher energy photons cause photodissociation to compete against fluorescence (654).The resonance fluorescence of nitric oxide a t 215 nm, excited by sunlight, was used to measure the NO concentration a t many points in the upper atmosphere from a n orbiting satellite (55), and the results were utilized to study atmospheric transport processes ( 762). The fluorescence yield of nitrogen dioxide a t 25" was measured a t 486, 558, and 630 nm, under excitation a t 405, 436, and 480 nm, and quenching effects by foreign gases were evaluated (96).For carbon monoxide analyses in the atmosphere, its infrared fluorescence was employed in a non-dispersive infrared analyzer which has a sensitivity of 1 ppm and good specificity (586).The fluorescence spectrum of hydrogen excited by extreme-ultraviolet solar radiation was observed during a purge of the Apollo 17 hydrogen fuel cells (219).The absorption and fluorescence spectra of both normal liquid helium and superfluid helium excited by electron-beam bombardment, have been discussed (229). Frozen solutions in 4 to 9 N HC1 or HBr are suitable for the fluorometric determination of As, Sb, P b , T1, Bi, Se, and Te a t -196", according to a Russian review which also describes the techniques needed (817).The same workers have specified optimum conditions and excitation and emission peaks for cryogenic fluorometry of Te, P b , and Bi in 4.5N HC1, with good linearity and a sensitivity of 0.1 wg ml(40). i n the more usual field of liquid solutions, several papers discussed the fluorescence of a given reagent with several different metal ions. The quantum yields of the Be, Al, Sc, Ga, and Zr complexes with 2,3-dihydroxynaphthoic acid (21) and with chromotropic acid (17) were reported. In the semiquantitative determination of Li, Mg, Sr, Ca, Zn, Pb, and La with morin, higher sensitivity was achieved by concentrating the solution with a ringoven (261).Fluorometric analysis for Fe, Cd, Co, Mn, Cu, Ni, Hg, Ag, Pb, and Zn was studied by forming the phenanthroline complex of the metal and then a n ion pair of this complex with the anion of a hydroxyxanthene dye such as eosin or erythrosine; good success was achieved for Co, Cu, and Zn, by a procedure of extracting the ion pair from p H 9 aqueous phase into chloroform and dilution with acetone (535).In an effort to develop better fluorometric reagents based on o, o'-dihydroxyazobenzene for the determination of Al, Ga, In, Sc, and Mg, 11 derivatives of varied molecular structure were prepared; sulfonic acid and p-methoxy structures showed increased fluorescence, but the widely-used Superchrome Garnet Y and Lumogallion seemed to be excellent for aluminum and gallium (357). A study of oxine complexes of 17 metals gives the emission and excitation spectra and fluorescence lifetimes and quantum yields, and considers the possible use of the data (which show systematic effects of atomic number) for kinetic analysis of metal ion mixtures (554).Luminescence and other properties were described for the complexes of divalent Ni, Co, Mn, Zn, and Cd thiocyanates with some imidazole compounds (254).An investigation of the Be, Al, Zn, Ga, In, and Sc complexes of N-(salicy1idene)-ohydroxybenzylamine showed this reagent to be suitable for fluorometric determination of Zn, Be, and A1 down to about 50 ng (372).Salicylaldehyde 2-quinolylhydrazone and other heterocyclic hydrazones of o-hydroxyaldehydes
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were found to be very sensitive fluorescence reagents for detection of Zn, Al, Sc, Ga, and In, as well as suitable for quantitation of, for example, Zn a t p H 6.9-7.2 in 80% EtOH (845). Acridine fluorescence quenching by iodine was proposed for the indirect determination of Au, Cr, Se, and Te (866). The quasiline absorption and fluorescence spectra of porphine and its Zn, Cu, and Ni complexes were observed a t 77 OK in an epoxy matrix (82), and similar low temperature studies were done on vanadyl complexes of porphyrins of biological origin, in hydrocarbon matrices (289). Fluorescence and phosphorescence yields, and phosphorescence lifetimes, were reported for a series of porphyrin complexes containing Si, Ge, Sn, or Pb (286). The characterization of polymers by their trace metal impurities was facilitated by such fluorometric reagents as oxine for A1 and stilbexon for Fe (608). Aluminum, Beryllium, Boron. The classic Pontachrome Blue Black azo dye reagent was used in the fluorometric determination of aluminum in biological materials a t the microgram level with high specificity (724). Submicrogram amounts of aluminum in cesium halides or sodium iodide were determined by the fluorescence of the salicylidene-o-aminophenol complex (212). The same reagent served in analyzing NaI or Mohr's salt for traces of aluminum, in a method which extracts the fluorescent complex with 2M tributyl phosphate in chloroform, from an aqueous phase a t p H 6 and 3M in sodium perchlorate (170). N-salicylidene-o-aminophenylarsonicacid forms a 1:1 neutral complex with aluminum a t pH 4-5 which has peak emission a t 545 nm and provides 0.1 rg/ml sensitivity (192). Nonionic surfactants were found to enhance the sensitivity remarkably, in the fluorometry of aluminum complexes (389). The determination of aluminum in ferrotitanium was done by fluoride titration with the fluorescent indicator o-Cresolphthalexon S after complexing the titanium with ascorbate (442). Aluminum sulfate residual catalyst in polydimethylsiloxane rubber can be quantitated by the severalfold increase in luminescence intensity which results from heating in argon a t 300" for an hour (721). Superchrome Garnet Y continues to be used for the determination of aluminum and gallium, a t pH 5 and 3, respectively; scandium and indium also form fluorescent complexes, but Fe, Co, Cu, Zr, V, and Cr negatively interfere (358). Aluminum, gallium, and scandium form strongly fluorescent complexes with the new reagent C.I. Mordant Blue 31 at pH 5-7, 4, and 6, respectively, and having emission peaks a t 590, 600, and 610 nm, respectively (359); Y, La, Lu, In, and Mg also give fluorescent complexes with this reagent in acidic or basic medium. For the fluorometric determination of beryllium, complexation with o-(salicy1ideneamino)phenylarsonic acid a t pH 6 was found to give a sensitivity of 1 ng/ml (887) and to be free of interferences by many other metals (884). Another procedure for Be uses 6-methyl-1-hydroxyxanthone to form a fluorescent complex in 1:l methanolwater containing p H 8 buffer; the complex is extracted with cc14 which is then dried thoroughly, and the fluorescence measured a t 470 nm with 435 nm excitation (624). Of 13 derivatives of 5-hydroxychromone tested as fluorometric reagents for Be, the 2-ethyl-3-methyl- and the 2methyl-7-methoxy-5-hydroxychomoneswere judged suitable, and the fluorescence was found to be most intense in
cc14 (391). The fluorescence produced by 4'-chloro-2-hydroxy-4methoxybenzophenone (CHMB) in 90% HzS04 has been used to determine boron in natural waters (613) and blood (614) in the 1 ng/ml range; the intensity a t 490 nm shows a linear calibration curve. In connection with this method, details have been given for the quantitative separation of B as trimethyl borate by isothermal distillation (506). An automated CHMB procedure has been developed for boron in waters, detergents, and sewage effluents, in which interferences have been removed (9). Carminic acid was used for determining B by the fluorescence intensity a t 585 nm with excitation a t 476 nm a t pH 7 (101). The fluorescence of the B complex with salicyclic acid in 2:3 sulfuric-acetic acids was intensified tenfold by lowering the temperature to - 196" (704). Another sensitive fluorometric method for B in water, soil, plants, etc., used the borosalicylate complex to form a fluorescent ion pair with 502R
Rhodamine 6G which was extracted into benzene (949). For the fluorometric determination of submicrogram amounts of boron in high-purity phosphorus, resacetophenone was preferred to 2,4-dihydroxybenzophenonebecause of its greater stability even if lesser sensitivity (491). Calcium, Strontium, Magnesium, Lit'hium, Sodium. Details were given for determining calcium down to 1 ppb in water or ethanol, by the green fluorescence of its complex with 8-hydroxyquinaldinealdehyde 8-quinolylhydrazone in 0.1N KOH; Sr, Ba, and Mg in moderate excess did not interfere, but many other metals caused decreased intensity (217). The same reagent was utilized to determine small traces of Ca in cesium chloride (90). Great interest continues in the jellyfish protein aequorin as a luminescence reagent for simple low-cost analysis of calcium, for example in clinical chemistry where a linear calibration curve was obtained on logarithmic coordinates (394). Directions for preparing the reagent from Aequorea jellyfish circumoral rings were given, and the light yield a t 25" was found to be 4.5 x 1015 photons per mg of aequorin, with excess Ca (408). Two kinetic studies of the binding were reported, one of which discussed also the application to intracellular localization of Ca (341) and the other found S r also to be effective in producing light, although a t much lower intensity (544). Light is produced with aequorin not only by Ca and Sr, but also by more than 10 other cations (395); La, Y, Pb, and Cd all gave considerable luminescence a t pH 6 but less a t pH 8 (828). Chlorotetracycline has been used as a fluorescence reagent for biological in situ Ca analysis in nerve membranes (325), isolated sarcoplasmic reticulum from skeletal and cardiac muscle (128), and the inner membrane of mitochondria (127). Fluorescent indicator titrations of calcium with EDTA were reported using a potassium-calcium salt of calcein as the indicator (383) and in KC1 and NaCl single crystals, using fluorexon indicator (411). In the fluorometric determination of Ca with calcein, optimum conditions were established for the best linearity and reproducibility (49). Lumomagneson, which is a Na salt of 2-hydroxy-3-sulfo4-chlorophenylazo-1-barbituricacid, was used as a fluorescent marker for mineral deposits in bones, but could not distinguish between Mg and Ca (146). Another fluorescence reagent for both Mg .and Ca is l-dicarboxymethyl-aminomethyl-2-hydroxy-3-naphthoic acid (DHNA) which can be used either as an indicator in chelatometric titration with EDTA or else for detection of Ca, Mg, Zn, Cd, Sr, Al, or T h a t a sensitivity of about 1 pg/ml (629). Sensitive fluorometric procedures for magnesium were published using morin a t pH 9.85 in aqueous methanolethanol (475), 3-hydroxy-3',4'-dimethoxyflavone, with elimination of interferences found in urine and serum by strong-acid exchange resin (345), and bis(salicylalethy1enediamine) in anhydrous dimethylformamide containing isobutylamine (92). The 1:l fluorescent complex of lithium with oxine was found to be largely covalent, in contrast to the ionic salts formed by other alkali metals (792). Sodium hydroxide and sodium carbonate in electrolysis brine were determined fluorometrically with o-phenylphenol (386). Elemental sodium vapor was detected a t levels down to 1.6 x 1 O - l ' gram by its resonance fluorescence in the gas phase under laser illumination a t 589.6 nm (404). Copper, Silver. Copper in alloys and microgram amounts of Cu(I1) in solution were detected by the yellow fluorescence produced on treatment with pyridinium iodide in ethanol; the fluorescence changed reversibly to red and violet upon cooling to -180" and -196", respectively (337). Thermochromism was shown also by some of the fluorescent complexes of cuprous halides with alkylpyridines or aminopyridines (333, 335) and with heterocyclic bases such as quinolines or nicotinic acids (334). Fluorescence and other photometric methods for silver were reviewed in a Russian paper with 119 references (536). The quenching of eosin fluorescence a t pH 4 was used t o determine Ag and P b in microgram amounts (883); eosin was more sensitive than fluorescein for this purpose, and the method was more sensitive for lead than for silver (888).A fluorescence procedure for silver in ores includes preliminary separations and makes use of the ion
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pair of eosin with the Ag complex of 1,lO-phenanthroline (821).
Bismuth, Lead, Zinc, Cadmium, Mercury. Submicrogram amounts of bismuth were determined by the solidstate fluorescence of CaOaBi crystals a t -196", and optimum conditions and the effects of other metals ascertained (213, 456). Another paper on the same subject recommends 345-65 nm excitation and 404 nm emission for the quasilinear luminescence spectra, and points out t h a t lead can be determined simultaneously by excitation of the C a O - P b solid a t 336-40 nm and measurement of the fluorescence a t 365 nm (947). Further details on P b analyses by this method were published (455). A different fluorometric sensitive procedure for P b involves recording the changes in 480-520 nm emission upon heating a frozen HC1 solution containing lead (841). Excitation and emission spectra, as well as quantum yields, were reported for P b in germanate, borate, and phosphate glasses (741). Zinc in ppb amounts was analyzed by the 520-nm fluorescence (under 470-nm excitation) of its complex with benzimidazolecarboxaldehyde 8-quinolylhydrazone ( 766). Less sensitivity for Zn was shown by P-(salicylideneamino)-ethanol in acetate buffer, with 375-nm excitation and 440-nm emission; some positive and negative interferences were listed (435). The fluorescent complexes of Zn, Cd, and Mg with N-salicylidene-4-aminobenzothiazoleform optimally a t pH 8.9, 10.7 and 8.7, respectively, and can be extracted with isoamyl alcohol and used for quantitation, especially of Zn in the ng/ml range (827). Salicylaldithiocarbazinic acid esters also form fluorescent complexes a t p H 8-10 with Zn, 365-nm excitation giving 480-nm emission, which are useful for Zn analysis in the pg/ml region (431). Fluorescent complexing of zinc with 8-hydroxyquinoline-5-sulfonic acid makes possible either the direct titration of Zn with this reagent, or else its use as fluorescent indicator in the EDTA titration of Zn, a t concentrations above 10-6M (943). Hg(I1) is readily determined in submicrogram amounts by its oxidation of thiamine a t pH 9.5 to strongly fluorescent thiochrome, but foreign salts and many other ions interfere (370, 1007). A fluorometric microdetermination of cadmium employed 8-p-tosylaminoquinoline as the reagent, a pH 11 buffer, and measurement a t 515 nm (954). Gallium, Indium, Thallium. A comparative study of gallium fluorescence and color reactions with lumogallion, pentahydroxyflavone, oxine, rhodamine C, and o-(salicylideneamino)phenol was reported by Matveets and Shcherbov (583); the same authors gave detailed data for fluorometric determination of Ga with o-(salicylideneamin0)phenol (585) and with sulfonaphtholazoresorcinol (584). A procedure was presented for the analysis of nanograms of Ga in biological material, using the fluorescent complex with lumogallion as extracted into isoamyl alcohol (1025). Other complexing reagents utilized in fluorometric methods for Ga include Acridine Orange (297), 2-hydroxy-lnaphthaldehyde thiosemicarbazone (434), Superchrome Garnet Y (358), Mordant Blue 31 (359), and 4-salicylidene-aminoantipyrine (886). Both gallium and indium form complexes with lumomagneson which fluoresce a t 521 nm, but many ions interfere (14). Excitation spectra for the luminescence of aqueous solutions of LiCl and LiBr containing thallous ion were given (63), as well as excitation and emission spectra for T1+ in aqueous solutions of ammonium chloride and the mono-, di-, and triethylammonium chlorides (568). Iron, Chromium, Rhenium, P l a t i n u m Metals. Tetrachloroferrate(II1) compounds a t 85 O K showed a broad luminescence band in the near infrared when excited with blue or ultraviolet radiation (233). For the fluorometric analysis of chromium(II1) as complexed with thiocyanate and extracted into tributyl phosphate, the emission intensity increased and the peak shifted t o deeper red as the number of NCS- groups in the complex increased (906). In dimethyl sulfoxide solution, the Cr(II1) thiocyanate complexes show phosphorescence, the spectra of which were studied over the range 77-150 "K (500). Fluorescence quenching methods for Cr(II1) were described, using, for example, the decrease in emission a t pH 5 of the Zn complex of salicylaldehyde acetylhydrazone (373). Rhenium(VII1) was quantitated by treatment with Acridine Orange in aqueous H3P04 and extraction of the 520-
nm fluorescent complex into dichloroethane; microgram amounts in Cu-Mo ores were determined (296). In the fluorometric analysis of ruthenium(I1) as its 1:3 complex with 1,lO-phenanthroline, serious interference by Fe, Co, Ni, and Bi was found but procedures to remove them were developed (818). A catalytic reduction of Ce(1V) to fluorescent Ce(II1) by iridium was used in a method for milligram amounts of iridium, and the interferences by other metals were noted (819). Absorption and emission spectra for mixed chelates of rhodium(II1) with 1,lO-phenanthroline and 2,2'-dipyridyl were reported (326). The rhodium and palladium complexes with phthalocyanine were found to fluoresce as well as phosphoresce in dilute solution (601).
Germanium, Antimony, Tin. Fluorometric and other methods for germanium are the subject of a Russian review, with 100 references (822). A luminescence determination of nanogram quantities of antimony in sodium iodide is based on the red emission a t 640 nm of an Sb(V) solution in HBr a t -196" (842). Morin was used for the fluorometric determination of both S b and S n in minerals, by differential extraction of the complexes and re-extraction from benzene into 0.05N and 0.2N HC1, respectively (815). Small percentages of tin in light alloys were measured by the 580-nm fluorescence of the benzene extract of the tin-Rhodamine B complex (645). Zirconium, Hafnium, Thorium. Optimum conditions for the fluorometric determination of Zr as its 1:2 complex with salicylidene-4-aminoantipyrine were: 366-nm excitation, 554-nm emission, 40% E t O H solution containing HCl ( p H 1-2), threefold excess of reagent, sensitivity 8 ng ml (892); it was possible to mask interferences by Hf, Ti, L o , W, T h , Pb, Fe, Co, and Ni, and a linear calibration curve was obtained (885). Improvements were reported for the spectrofluorometric method for hafnium in the presence of zirconium using quercetin, a t 396-nm excitation and 498nm emission (479); the effects of different acids and different solvents on the fluorescence of Hf-quercetin complexes were investigated (687),and optimum conditions were specified (498). A paper chromatography method for Hf includes separation of Zr and Ti, and measures the fluorescence after treatment with quercetin (278). Ethanol and other organic solvents were found to increase the sensitivity of fluorometric determination of Hf with morin, and preliminary separation of Zr was achieved by a cation exchanger (688). Morin was also used as a fluorescence indicator in the complexometric titration of microgram amounts of thorium with Complexon I11 or oxalic acid a t pH 1-3, with good sensitivity and specificity (867); the method was applied to the determination of T h in tungsten wire, after separation as the fluoride (865). Niobium, T a n t a l u m , Vanadium, Uranium. Pilipenko and coworkers have studied the absorption and emission characteristics of niobium complexes with lumogallion (691) and sulfonaphtholazoresorcinol (694) in the presence of auxiliary complexers such as H202, fluoride, tartrate, and oxalate. They found increased sensitivity in the presence of H202 and organic solvents (methanol) and specified optimum conditions for these reactions and also for the fluorometric determination of niobium with quercetin and H202 a t 500 nm, with 436-nm excitation (693). A comparison of various reagents for N b indicated the fluorometric determination with lumogallion a t low temperatures as promising (816). For the fluorometric determination of tantalum in the presence of H202, the most sensitive reagent was found to be Rhodamine 6G, followed by Butylrhodamine S, morin, sulfonaphtholazoresorcinol, and lumogallion in that order (692). A simple fluorometric method for vanadium in the ppb range uses the reaction with benzoic acid in the presence of zinc amalgam; Fe(II1) and Ti(1V) interfere, but many common ions do not (467). Sharp line luminescence in the near infrared was observed for solid perchlorate and iodide salts of the V(urea)e3+ ion a t cryogenic temperatures (232). Separation procedures are included in many of the papers which deal with the fluorometric determination of uranium: in natural waters by the fluorescence in sulfuric -phosphoric acid solution (158); in urine (474); in sea water, by the 575-nm fluorescence of the rhodamine B complex (524); in high-purity silicon dioxide (175); and in sodium iodide, by the fluorescence of the fusion bead of
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sodium fluoride and carbonates (567). In the fluorescent bead method, there must be careful control of such experimental variables as composition of flux, fusion temperature and time, fusion atmosphere, and elapsed time between fusion and measurement (13, 58). Other fluorescence procedures for uranium involve its quenching of the emission a t p H 9-10 of N-(8-hydroxyethy1)anabasine (563) and oxidation titration by Ce(1V) after passage through a Jones reductor, with detection of the end point by the fluorescence of either U(VI) or Ce(II1) (470). The dependence of emission intensity on uranium concentration in HzS04 solutions was measured, under electron-beam excitation (282). Scandium, Rare Earths. Anisic acid P-hydroxynaphthalhydrazide gives a yellow-green fluorescence with scandium a t p H 2.0-3.5 in 1:l acetone-water, which was used to analyze for submicrogram amounts of S c in rare earth oxides; rare earths do not interfere, but some other ions do (176). The intrinsic fluorescence a t 350 nm of Ce(II1) in 0.5N HzS04 was utilized in a fluorometric method (820). Optimum conditions were ascertained for the emission of the morin complexes of the rare earths, and the quantum yields were measured (385). Of the phenylpropiolic acid complexes of rare earths, the strongest fluorescence was shown by E u (red) and T b (yellow) (420). The fluorescence reaction for lanthanum with bis(4hydroxybenzoylhydrazones) of a-diketones such as glyoxal was found quite sensitive (526). Studies have been reported on fluorescent complexes of europium(II1) with nitrosalicylaldehydes (501) and with dibenzoylmethane (45, 250, 471), as well as of E u and Gd complexes with 1,lO-phenanthroline and thenoyltrifluoroacetone (807). A fluorometric procedure for gadolinium is based on the 571-nm emission (with excitation at 546 nm) of its complex with Rhodamine S and salicylate (686). The fluorescence of Gd ion in solution increased significantly upon complexing with citric, tartaric, pyrophosphoric, or several other acids (711). In the spectrofluorometric determination of terbium as its 1:l complex with a,a’-ethylenediiminodi(0-hydroxyphenylaceticacid) a t 545 nm, a linear relation was obtained down to 1.5 ng/ ml, but Th interfered seriously (882). Terbium forms a strongly fluorescent complex with pyrogallolsulfonic acid also, but it is destroyed as the UV irradiation continues; some of the rare earths do not interfere, but others do quench the fluorescence (708). Both terbium and dysprosium form 1:1:1 complexes with EDTA and 1,2-dihydroxybenzene-3,5-disulfonic acid a t p H 12-13 which fluoresce at 546 and 572 nm, respectively, and are good for subnanogram analyses (908). Sensitive fluorometric methods for T b , Eu, and S m were developed, using the hexafluoroacetylacetone complexes (993). Papers continue to appear on the great enhancement of fluorescence intensity attainable in POC13-SnC14 solvent, as compared to aqueous solution, for E u (68), S m (910), and Nd (29); for example, a n enhancement factor of 220 was found for Eu, and a factor of 300 was found for S m in the related “heavy atom effect” solvent POC13-ZrC14 (136). The measurement of luminescence of solids, as a n analytical method, has been applied to rare earths widely (430), using excitation not only by ultraviolet but also by X-rays (734) or 700 keV protons (31). With YVo4 as matrix, this technique was employed to determine Gd, Eu, and S m in metallic uranium (28), Eu and Gd in geochemical samples (27), and S m and E u in neodymium oxide (709). Similar procedures were reported for trace analysis of praseodymium in yttrium oxide (710), of europium in yttrium oxysulfide phosphor (664), and of T b , Dy, and Ho in high purity lanthanum oxide (198). For the fluorometric determination of thulium in borate and phosphate glasses, a linear relation was obtained between T m concentration and the intensity of the 454-nm line, using 358-nm excitation (739). The fluorescence quantum efficiency was measured for Eu(II1) in phosphate glasses (742).
Nonmetals. Analytical methods for selenium were reviewed, with the conclusion that the best for low concentrations is fluorometry of the 2,3-diaminonaphthalene complex (669). This complex, piazselenol, was extracted into cyclohexane and the emission measured a t 520-530 nm, in procedures for determining Se in lake sediments 504R
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(990), ores and minerals (640), and highly pure arsenic trichloride (546). Selenium and tellurium were determined in very pure arsenic, antimony, and their compounds by the luminescence a t 77 “K of piazselenol and of Te(1V) in frozen 9 N HC1, respectively (547). Another method for Te(1V) is fluorometry of its rhodamine dye complex obtained by benzene extraction from 2M HBr solution (454). Trace amounts of sulfide were determined fluorometrically by quenching titration with fluorescein tetramercuracetate (91) or else indirectly, by adding a n excess of standard cupric solution and measuring the quenching of 2-(o-hydroxyphenyl)benzoxazole fluorescence caused by the excess Cu (953). For sulfate in natural waters, quenching of the thorium-morin complex fluorescence was a sensitive method, but subject to interference by phosphate, fluoride, several cations, and some organic compounds (639, 958). Nitrite in nanogram amounts was measured by its diazotization of p-chloroaniline, followed by coupling with 2,6-diaminopyridine and further derivatization by ammoniacal CuSO4 to give a highly fluorescent triazole compound, having 360-nm excitation and 430-nm emission (177). Other methods for nitrite used the fluorescence reaction with Ethylrhodamine S (700) or the intensity decrease and wavelength shift of the fluorescence of benzidine (662). Phosphate was determined a t the ppm level by the 445-nm fluorescence (352-nm excitation) of a quininemolybdophosphate complex in 9: 1 acetone-0.5M (459), and a t ppb levels by reaction of the molybdophosphate complex with thiamine to produce the highly fluorescent thiochrome (371). Fluorescence methods reported for quantitation of cyanide include: an indirect procedure, as noted above for sulfide, involving excess Cu and its quenching of 2-(o-hydroxyphenyl)benzoxazole (953); liberation of piazselenol from its nonfluorescent complex with PdC12 (593); and the use of copper-calcein or leucofluorescein as reagents (765). A highly sensitive procedure for iodine, as in rocks, is fluorometry of its complexes with rhodamine dyes which form in strong acid solutions in presence of Br- and are readily extractabie by benzene (701, 702). Oxygen concentrations were measured by the quenching effect on pyrene (851, 861) or, for intracellular measurements, on pyrenebutyric acid (464); oxygen quenching of protein fluorescence was used to study rapid structural fluctuations in proteins (504, 505). Chemiluminescence Analysis. Methods for the determination of osmium(VII1) (98), copper(I1) ( 1 8 9 , and chromium(I1) (188) were based on the changes they cause in the chemiluminescence of lucigenin (10,lO’-dimethyl9,9’-biacridinium nitrate) in alkaline solution and presence of a n oxidant such as HzOz. Luminol is even more widely used for chemiluminescence analysis (805); recent inorganic applications have been to gold (689), iridium(II1,IV) and rhodium(II1) (690), iron(I1) (804), complex formation between cobalt and 2-nitroso-1-naphthol ( 5 5 4 , ozone (858),and hydrogen peroxide (189, 1024). Siloxene chemiluminescence in 6 M HzSO4 was linearly related to vanadium(V) concentration a t trace levels, but oxidants interfere and tungstate, molybdate, and several other ions quench the luminescence (295). Organic analysis procedures using luminol chemiluminescence included those for tricresyl phosphate in ethanol (859), triethylene glycol impurity in organic solvents (860),and several aromatic aldehydes (472). Luminol was proposed as a secondary standard light source in bioluminescence studies (633), and its luminescence characteristics in dimethyl sulfoxide (DMSO) and basic aqueous DMSO solution were measured (284); the mechanism of its light emission was studied by pulse radiolysis (59). Other papers discussed the chemiluminescence produced by autoxidation of dihydroxyphenylalanine (81), by the ozonization of 3-aminobenzaldehyde (644), and by the reaction of methyl radicals with oxygen in aqueous solution containing Ti(I1I) and tert-butyl peroxide (923).
ORGANIC AND BIOLOGICAL
A new fluorometric technique of general applicability is the use of solid surface reactions, in place of liquid re-
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agent solutions which deteriorate faster; the method consists of forming a solid film of one reactant on an inert silicone matrix pad, then contacting with the solution to be analyzed, and measuring the fluorescence (305, 307). Another interesting report describes the strong triplet-state phosphorescence a t room temperature shown by salts of polynuclear amines, acids, or phenols adsorbed on paper, silica, alumina, etc., with spectra similar to those in frozen solution a t -196" (788). A fluorometric fast analyzer was developed and applied to kinetic assay of enzymes and other determinations in clinical chemistry (905). The usefulness of the Brown cuvet for steroid and many other fluorometric analyses was pointed out; it can be used for extractions, centrifuging, and as a fluorometer cuvet (531).
Hydrocarbons a n d Heterocycles. An interesting innovation which may be important for the future is the use of vacuum ultraviolet excitation a t 147 and 165 nm to excite the characteristic fluorescence spectra of a wide variety of alkanes, cycloalkanes, and polycycloalkanes (760). Slow electron impact was also used to excite the luminescence of thin films of alkanes a t 77 "K (360). Long chain aliphatic hydrocarbons in airborne particulates were determined on silica-gel chromatography plates impregnated with Rhodamine 6G (1000). In the technical analysis of petroleum and related fields, fluorometric techniques were applied to: the determination of product quality of kerosine and lubricating oil (715); the characterization of high-boiling distillates (590); the fluorescent indicator adsorption method for analysis of paraffin-naphthene fractions (679) and gas oils (622); the detection of petroleum traces in sea water (444); the identification of naval fuels in sea water (382) and the remote sensing of oil spills a t sea by airborne-laser excitation of fluorescence (214); the forensic identification of automobile engine oils and gasolines (538-540); the nature of arene resins in crude oils and asphalts (48); the characterization of pyridine extracts of coal (744); the determination of the aromatic content of petroleums and bitumens (463, 823); and the study of weathering qualities of bitumens (346). Benzene and other simple aromatic molecules in solutions showed a decrease of fluorescence yields and lifetimes with temperature, and also solvent effects (151). Quasilinear phosphorescence spectra a t 77 "K were used for analysis of benzene a t ppm levels in subsurface waters (1021) and in a suggested analytical procedure for benzene and its alkyl homologs (429). Studies were reported on the fluorescent spectra and lifetimes of the first excited singlet state of benzene, as a function of temperature and of concentration in cyclohexane (152), on fluorescence from highly excited states of benzene, toluene, naphthalene, et al. in the vapor phase (293), and on fluorescence in solution from higher excited states of benzene and other aromatic hydrocarbons (361), including 3,4-benzopyrene and 1,12-benzoperyiene (124). Analyses for 3,4-benzopyrene continue to be of interest, because it is a carcinogen; papers have appeared on its determination by fluorometry in tea (829) and in the environment (451), in pure n-octane (216), and in petroleum paraffins for the food industry (453) by the quasiline emission a t 403 nm obtained a t -196". The quasilinear emission and excitation spectra a t this temperature were measured for several methyl and dimethyl derivatives of 3,4benzopyrene (665). Corrected fluorescence spectra for 20 polynuclear aromatic compounds were presented (716). A method for the determination of polynuclear aromatics in natural water utilized fluorescence and phosphorescence, after thin-layer chromatography (TLC) separations (200). A large number of polynuclear aromatics were found in coal tar and quantitated, using two-dimensional TLC followed by spectrofluorometry (581). Quasilinear luminescence spectra were employed for the simultaneous determination of eight polynuclear hydrocarbons including benzpyrenes (452), the study of the temperature and concentration dependence of coronene emission from frozen solutions in n-paraffins (2911, and the study of anthracene derivatives to find the effects of substituents as a basis for analytical methods (938). Other papers on aromatic hydrocarbons concern: the spectrofluorometric determination of traces of naphthacene (377); the fluorescence properties of naphtha-
cene in n-paraffin matrices a t 77 O K and 4 O K (1022); sensitized luminescence in thin layers of naphthacene in 1,2benzpyrene (378) and of naphthacene in phenanthrene (376); the fluorescence spectra of 1,12-benzoperylene in n-hexane and of perylene in n-octane over the range 4.280 O K (20); the fluorescence spectra changes caused by phase transitions of the frozen cyclohexane matrix (269, 270); and luminescence quenching by oxygen a t 77 "K (311) and by oxygen and nitric oxide (410). The phosphorescence spectra a t 77 OK were reported for phenanthrenequinone (499) and for frozen solutions of triphenylene, hexahydrotriphenylene, coronene, diphenylene oxide, and diphenylene sulfide in CCI4 and iso-octane, some of which were quasilinear (292). Phenanthrene and carbazole impurities in anthracene were determined by measuring the phosphorescence decay time a t 462 nm (752). A quasiline structure for the emission a t 77 "K was shown by 9,lO-diazaphenanthrene in hexane (57) and by azulene in pentane (657). Fluorescence yields a t room temperature were determined for cyclohexane solutions of 20 derivatives of azulene (626), many of which emitted fluorescence from the second excited state (625). Polarized fluorescence, phosphorescence, and absorption spectra were measured for 1,3-diazaazulene (110). In a series of benzocycloalkanes, the fluorescence yield tended to decrease with ring size (300). A systematic study of emission and absorption of phenylcyclopropanes and phenylethanes at 77 OK showed some unusual features, mechanisms for which were proposed (60). A ternary mixture of halogenated biphenyls was analyzed quantitatively by pulsed-source time-resolved phosphorimetry (649). For biphenyl and 9-chloro derivatives in ether-isopentane-alcohol (EPA) glass, the emission spectra and quantum yields were measured and correlated with molecular structure (185). Similar studies were done on the effect of chlorine and oxygen atoms in benzene, naphthalene, and biphenyl derivatives on their phosphorescence spectra (895) and on heavy-atom effect (due to use of propyl chloride, bromide, or iodide as matrix) on the phosphoresceme spectra of halonaphthalenes (903). Many reports appeared on the luminescence of heterocyclic compounds, such as: the changes in fluorescence of carbazole and N-ethylcarbazole over the p H 1-14 range in aqueous solution (121); the phosphorescence of 1,5-naphthyridine (227); the very intense fluorescence of some 2,4,6-trisubstituted pyridines and their potential use in liquid scintillation counting (165, 722); the phosphorescence and excitation spectra of pyrazine species in ethanol-water solutions (576); fluorescence of the cinnolines in solution (863); the quasiline emission spectra a t 77 OK of solutions of several methylquinolines (400), 5,6- and 7,8benzoquinolines (401), 2-phenylbenzothiazole and 2-phenylbenzoxazole (656), 1,4-benzodioxane (1027j, anthraselenadiazole (494), naphthoxadiazole and naphthothiadiazole (495), anthrathiadiazole (496), and anthraoxadiazole (497); and the fluorescence properties of indolizines and some azaindolizines (523). Other papers discussed the phosphorescence of benzoylthiophene derivatives (35) and photochromic spiropyrans (47), and the fluorescence of benzimidazoles as affected by pH (749), of aromatic benzoxazoles (736), and of 2-substituted benzothiazoles in terms of the use of o-aminothiophenol as a fluorometric reagent (636). Oxygenated Molecules. Of the' 66 aldehydes and ketones tested, nearly all of the aromatic aldehydes promise to be suitable for fluorometric determination in the microgram range by condensation with o-aminothiophenol to form phenylbenzothiazoles; procedural details are given for vanillin (933) and for furfural and sugars (635). Acrolein was analyzed by the fluorescence of its condensation product with m-phenylenediamine in acid medium (388). Turro et al. reported that, contrary to expectations, it was possible to observe the phosphorescence of acetone in organic solvents, excited a t 313 nm, by nitrogen purging of the solution to remove quenching by oxygen (924). The fluorescence and phosphorescence spectra of the isomeric naphthaldehydes and naphthyl methyl ketones were studied in hexane and aqueous HzS04 solutions (481). Benzaldehyde and acetophenone showed phosphorescence and intense fluorescence in HzSOa, due to their protonated species (673). For the isomeric hydroxybenzaldehydes, the
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luminescence and absorption spectra were measured and compared with theoretical predictions (555). Benzophenone was the subject of several papers on: its prompt fluorescence, E-type delayed fluorescence, and phosphorescence in organic solvents produced by laser excitation a t 337 nm (100);its phosphorescence in aqueous solution (19); and its quasiline emission spectrum which ?as obtained by slow cooling of a toluene solution to 4.2 K (38, 84). Phosphorescence and absorption spectra of benzophenone and several derivatives in ethanol-water a t 77 "K were measured, in a study on hydrogen-bonded species (564). T h e quasilinear phosphorescence of anthrone in hexane was examined (38). Benzanthrone as an atmospheric pollutant was determined by its 465-nm fluorescence intensity, at -196", after TLC separations (396). A dependence on the viscosity of the glass matrix was found for the phosphorescence spectra of 1-ienzoylnaphthalenes (350)! Absorption and emission characteristics were measured for a group of aromatic carbonyl compounds (222). A series of rigid cyclopentenones was studied by recording phosphorescence spectra at 77 "K (122). Improvements were reported in the fluorometric determination of nanogram amounts of glycerol (from glycerides) by periodate oxidation to formaldehyde, which was then condensed with NH3 and acetylacetone to yield intensely fluorescent 3,5-diacetyl-1,4-dihydrolutidine (275). TLC procedures were described for triglycerides, which became fluorescent when heated a t 150" on the silica gel plates containing ammonium sulfate (915) and for phospholipids, where the spots were visualized with l-anilino8-naphthalene sulfonate (352). Lipid peroxidation products in biological systems, as a sign of damage, were assayed by their fluorescence characteristics (232). Glucose in blood or other biological fluids was determined fluorometrically by its reaction with 5-hydroxy-ltetralone in presence of CuSO4 (652), and by a lanthanum chelate of its reaction product with p-hydroxybenzoic acid hydrazide in diethanolamine solution (527). Automated routine analysis for glucose in clinical chemistry, using a fluorometric hexokinase procedure, proved satisfactory in more than 100,000 runs (154).A sensitive detection method for reducing sugars on paper chromatograms consisted of spraying with ethylenediamine sulfate and heating to develop the fluorescence (374). Trace impurities in commercial sugars were measured better by fluorescence than by color (968).For the fluorometric microdetermination of glycogen, one procedure used anthrone in sulfuric acid as the reagent (619) and another was based on consecutive enzymic conversions, with measurement of the amount of reduced dinucleotide coenzyme produced in the final step (631).Anthrone was utilized for the fluorescence analysis of pentoses also, and the optimum conditions were ascertained (362).Hydroxybiphenyls, as metabolites of biphenyl, were quantitated by the yellow fluorescence of their DANSYL derivatives (246). Changes in the emission spectra of 1-naphthol a t lower temperatures were reported (876). Enzymic fluorescence methods for analysis of samples of blood cells or blood were reported for 1,3-diphosphoglycerate (783),galactose 1-phosphate (155), several glycolytic intermediates and pyridine nucleotides (801), and 6 hydroxybutyrate (69). Citric acid was determined by the intense fluorescence which resulted from heating with resorcinol and HzSO4 (1005), or which resulted from treatment with KzC03 in acetic acid-acetic anhydride (507); aconitic acid behaved similarly toward the latter reagent. Glucuronolactone and glucuronic acid were analyzed simultaneously by fluorometry a t 457 nm after treatment with hydrazine and zirconyl chloride, before and after heating a t 70" (417, 428). In a procedure for microgram amounts of D-glucaric acid in serum, periodate oxidation gave glyoxylic acid which was condensed with 4'-hydrazino-2-stilbazole to form a compound which fluoresced strongly a t 550 nm, with excitation a t 450 nm (580). Aromatic acids in nanogram amounts were identified in situ after paper electrophoresis by means of their intrinsic fluorescence (964).In the fluorometric assay of homovanillic acid in urine by ferricyanide oxidation, it was necessary to remove interferences (460) as by means of a cation exchanger (425). A simple method for quantitating 3,4dihydroxyphenylacetic and 3-methoxy-4-hydroxyphenyla506R
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cetic acids in urine employed a combination of TLC and fluorometry (956).Interfering substances required preliminary removal before fluorescence analysis of 5-hydroxyindoleacetic acid in cerebrospinal fluid (991). (See also the section on Amino Acids.) Phosphorescence and excitation spectra of cinnamic acid a t 77 "K were reported (917 ) . Salicylate in blood was determined in simplified ways by using a blank of blood taken from the same subject prior to ingestion (650) or by using magnesium acetate to shift the excitation spectrum so that glass cuvettes could be used in place of silica (532).A new procedure for salicylate esters was based on their strong fluorescence in dimethylformamide solution containing a little K O H (416). For the fluorometric analysis of acetylsalicylic acid, the effect of added aliphatic acids was studied in order to find optimum conditions (781), and interference by salicylic acid and its conjugates was eliminated by oxidation with ceric ammonium nitrate (423). Nitrilotriacetic acid was determined by its quenching of the fluorescence of gallium-oxine complex (746). Uric acid was quantitated by coupled enzymic reactions producing H202, which caused a reduction in light emission by a n intensely fluorescent dihydrolutidine compound (276); and cyanuric acid was assayed by adding 4,4'-diaminostilbene-2,2'-disulfonic acid and measuring the fluorescence intensity (681). Coumarins and furocoumarins were identified in citrus oils by means of fluorescence profiles; the results were used to characterize oils of orange, lemon, lime, and grapefruit (575). Emission spectra of coumarins in proton transferring solvents were examined, in a study on hydrogen bonding (733). Phosphorescence was found to be stronger than fluorescence in several coumarins and psoralens which are skin sensitizers (573).Fluorescence shifts of 4-methylumbelliferone were studied as a function of the acidity of the solution (637, 1002). The emission spectra of fluoran and several methyl derivatives in absolute ethanol and 80% HzS04 were reported (299). A group of papers described the luminescence spectra, usually a t liquid nitrogen as well as room temperatures, of P-alkylanthraquinones (814), methoxyanthraquinones ( I I ) , polyhalo derivatives of anthraquinone (813, 925), and tetramethyl-pbenzoquinone (352). Another set of articles reported on the excitation and fluorescence spectra, as a function of acidity, of 8-hydroxyquinoline and 29 of its derivatives ( 9 n , chelating quinolinecarboxylic acids and their methyl esters (482), and cinchonic acid, cincophen, and their methyl esters (1026). Vitamins. In a survey of vitamins for luminescence analysis possibilities, it was found that p-aminobenzoic acid, folic acid, pyridoxine hydrochloride, a-tocopherol, calciferol, riboflavine, and retinol gave useful fluorescence, while the first four plus niacinamide gave useful phosphorescence ( 4 ) . Vitamins B1, Bz, Bs, BIZ, C, nicotinic acid, and nicotinamide were separated and identified by TLC in presence of a luminescence indicator (897). The intrinsic fluorescence of vitamin A around 485 nm was used for its determination in dairy products (902), foods (204), and serum (424);separation procedures were given, and also a correction method for the presence of phytofluene, based on the emission constants of pure retinol and phytofluene. Retinoic acid in hexane, when treated with 74% HzS04 and then diluted with water, gave a product fluorescing a t 423 nm (but very labile to light) whose intensity was a linear function of concentration a t microgram levels (392).The luminescence of /?-carotene in many different solvents was measured a t 77 "K and room temperature (609). Tocopherols in cerebrospinal fluid (950) and in foods, pharmaceuticals, and tissues (902) were quantitated by their native fluorescence. Phosphorescence analysis characteristics were obtained under various conditions for 1,4-naphthoquinone and sev,eral of the K vitamins (3,834, 922). Fluorometric procedures for thiamine in foods (6461, tissue culture media ( 6 0 3 , and urine (525),including a new automated method for urine analyses (675), continue to be based on the oxidation to thiochrome; many different schemes were used to minimize interferences. To determine added nicotinic acid in meat, it was converted on silica plates into a fluorescent compound by ultraviolet irradiation (104). Nanogram amounts of pyridoxal phos-
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phate in tissue were measured by the fluorescence of the and possible neuroregulator, which can be determined flucyanide reaction product (294), and 4-pyridoxic acid in orometrically by its intrinsic fluorescence a t 340 nm (332), urine ( a metabolite of vitamin B6) was determined after or a t 550 nm in 30% ethanol-1.5N HCI solution (159), or after condensation with ninhydrin (76) or with o-phthalalseparations by its own luminescence a t 440 nm (603).P a pers on direct fluorometric analyses for riboflavine (984) dehyde (148, 683, 727). Other metabolites are usually present such as 5-hydroxyindoleacetic acid (727) which suggest a photodecomposition-rate method (138) and discuss riboflavine decomposition products as studied by may interfere and therefore require separations or other TLC (315). For folic acid, the fluorescence and phosphoprecautions. Nanogram quantities of tryptamine were asrescence spectra were measured a t p H 5.6 and 10.5 (898), sayed by condensation with benzaldehyde followed by treatment with dry hydrogen chloride in ether solution to and a method was given for its fluorometric determination produce a fluorophore (537). For the determination of 11 in medicinals, including separations (714). catecholamine metabolites and tyrosine, phosphorimetry Amines. A major advance was the introduction of a new was recommended as superior to fluorometry in terms of reagent, Fluorescamine, for the fluorometric determinasensitivity and accuracy (848). tion of primary amines, amino acids, and proteins in the 10-12 mole range (929).This reagent behaves like a mixAmino Acids and Proteins. In continuation of the last ture of ninhydrin and phenylacetaldehyde used in a patparagraph, the amino acid tryptophan undergoes succesented analytical reaction for primary amines (928);it has sive metabolic transformations which give the biochemithe structure 4-phenylspiro[furan-2(3K),l’-phthalan]- cally important compounds 5-hydroxytryptophan or 53,3’-dione (978). HTP. 5-hydroxytryptamine or S-HT, and 5-hydroxyinIn blood and urine, primary and secondary amines such doleacetic acid or 5-HIAA. These three substances were as amphetamine were assayed by spectrofluorometry after assayed in brain and other tissues a t nanomole levels, by reaction with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole means of their intrinsic fluorescence in 3n’ HCI, after sep(612), and P-phenylethylamine was assayed after condenaration from each other (226); the same principle was sation with p-dimethylaminocinnamaldehyde (849). Aliused for 5-HIAA alone, in human urine ( 2 5 7 . An autophatic primary and secondary amines were determined by mated fluorometric determination of 5-HT and 5-HIAA the fluorescence of the cyclized product resulting from the was based on the o-phthalaldehyde-cysteine method reaction of their isothiourea derivatives with 9-isothiocy(473). Simultaneous assays of brain for 5-HT, 5-HIAA, anatoacridine (166).Long-chain amine salts, such as octacatechol amines. and homovanillic acid by fluorometry decylamine hydrochloride, were quantitated by luminesemployed o-phthalaldehyde for 5-HT and alkaline ferricycence titration with eosin a t p H 6.9 (489). Many nitrogeanide oxidation for homovanillic acid (343). Fluorometric nous bases including alkylamines and pyridines were deanalyses showed that the pattern of distribution of 5 tected by the fluorescence thermochromism of their spots HIAA corresponds to that of 5 - H T in biological samples on cuprous iodide test paper, observed by cooling in liquid (30). Related papers discussed: TLC determinations of nitrogen (336). S-HT, 5-HIAA, and other indoles, with enhancement of A sensitive procedure for nitrosamines involves reducthe native fluorescence by sprays containing H2S04 or dition to hydrazines, followed by condensation with 9-anthmethyl sulfoxide (314, 5.56): the simultaneous assay of’5raldehyde and 9-phenanthraldehyde to give highly fluoHIAA and normetanephrine (364) and the analysis of rescent hydrazones (1008).Cyclohexylamine in biological urine for another metabolite of tryptophan, namely 3-hysamples was measured after TLC separations by the fluodroxyanthranilic acid, by its native fluorescence under exrescence of its DANSYL derivative (879). A microdetercitation a t 350 nm (911,971). mination of acetylcholine was based on its intensification Another aromatic amino acid, tyrosine, undergoes a of the 540-nm fluorescence of a mixture of quercetin and physiologically important series of metabolic transformatetraphenyl hypoborate in absolute methanol (468).In the tions into 3,4-dihydroxyphenylalanineor dopa, dopamine. fluorometric assay of spermidine by condensation with onoradrenaline, and adrenaline. Fluorometric methods for phthalaldehyde, details were given for optimizing the sendopa and dopamine made use of ferricyanide oxidation to sitivity and avoiding interference by histamine (319). A the trihydroxyindole compounds (56, 966); the same propreferred method for histamine itself is a similar condencedure was used to determine dopa and.its derivative 3sation with o-phthalaldehyde, for which different opti0-methyldopa in the blood of Parkinson disease patients mum conditions have been described (25, 321, 830) and treated with dopa (153, 812), and also 5-S-cysteinyldopa which has been automated (206) and applied to wine (751). Melanin, the dark skin pigment of which dopa is a (699); preliminary separation of histamine from blood precursor, was assayed by the characteristic fluorescence samples was improved by use of a Dowex column (543). of its degradation products obtained by treatment with Catecholamines such as adrenaline and noradrenaline alkaline dilute HzOz (753). Melatonin, -Y-acetyl-5in biological materials continue to be an active area of methoxytryptamine, is a hormone which lightens skin fluorometric analysis (298, 308, 329): a popular method color by causing aggregation of melanin granules: it nlso uses mild oxidation with ferricyanide (140, 457) or iodine was determined fluorometrically in blood ( 1 4 1 ) . (684) to the fluorescent trihydroxyindoles, with stabilizaFor tryptophan itself, a method applicable to as small a tion of the intensity by a n antioxidant such as dimercapsample a s 0.01 ml of plasma was based on reaction with topropanol-formaldehyde (939). An automated version formaldehyde and then H202 to form a n intensely fluouses two fluorometers for differential determination of rescent norharman (992). The fluorescence. delayed fluoadrenaline and noradrenaline a t different wavelengths rescence, and phosphorescence spectra of tryptophan in (225).Other papers report a rapid determination of adrenethanol glass a t i7 K were studied (207).Tryptophan. tyaline in adrenal glands (33),a procedure using its intrinsic rosine, and phenylalanine fluorescence, both free and in emission a t 334 nm with excitation a t 281 nm. after ionproteins, was reviewed, and the fluorogenic reactions of pair separation (398),TLC methods involving fluorometry their monamine metabolites with HCHO were discussed of the spots after visualization with ferricyanide-ethylene(129). Formaldehyde was also used to induce strong fluodiamine spray (878) or as the DANSYL derivatives (918), rescence of tryptophyl peptides on silica gel TLC plates, and a histochemical procedure which uses condensation permitting the detection of submicrogram amounts if 9 5 ) . with gaseous glyoxylic acid to produce a fluorophore (43). Tryptophan metabolites in urine (kynurenine. 3-hydroxyFluorometric methods were described for 4- and 8-aminokynurenine, 3-hydroxyanthranilic acid. and xanthurenic quinoline, 8-hydroxyquinoline, and cinchoninic acid derivacid) were assayed by TLC and fluorometry (62). The atives (448), and luminescence characteristics of other photo-oxidation of tryptophan residues in proteins was aminoquinolines were measured under a variety of condishown by spectrofluorometry to produce ,\‘-formylkynuretions (790, 791). Luminescence spectra were also reported nine (696).Tyrosine residues in peptides were detected by for nitroanilines (450) and for LV,,V-dimethyl-p-cyanoanireaction with nitrosonaphthol to yield fluorescent line (449). benzphenoxazinone derivatives (320). The biogenesis of catecholamines and other amino compounds of physiological importance involves transformaPhenylalanine assay in blood or urine at the ultramicro tions of amino acids, so that there is overlap between this level as the basis of mass screening of infants for phenylsection and the following section. One interesting metaboketonuria has been reviewed 124). The preferred method lite is 5-hydroxytryptamine (serotonin), a vasoconstrictor employs ninhydrin and leucyl-alanine peptide as the reA N A L Y T I C A L C H E M I S T R Y , V O L . 46, NO. 5 , A P R I L 1974
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agents (369, 433); the same principle has been applied to ing of staphylococcal nuclease (203);alkaline phosphatase human granulocyte (26)and fish serum (595)samples. (259); transitions of the oxidized and reduced form of An automated fluorometric procedure for histidine in thioredoxin (368); native, denatured, and reduced-denablood (354) and a TLC method for histidyl peptides (194) tured proteins (492);binding of valyl-tRNA synthetase to made use of the fluorescence obtained by condensation valine-specific tRNA (349); binding of glucose to sweetwith o-phthalaldehyde. Histidine shows only a weak nasensitive protein from tongue epithelium (160); and the tive fluorescence, as observed a t neutral p H in aqueous binding of dodecyl sulfate to bovine serum albumin (324). solution (893). In the sensitive fluorometric determination Another technique was the use of external fluorescent of lysine by reaction with diacetylbenzene in the presence probes, which interact with the protein and provide inforof 2-mercaptoethanol a t p H 10, glycine and ornithine were mation about conformation; among the substances used found to give the same reaction (758). A test for arginine for this purpose were kynurenine (137), tetracyclines and other mono-substituted guanidines used phenanthrene(713), dimethylaminochalcone (847), and aminonaphthalene derivatives (891). Fluorescence polarization meaquinone as the reagent, and the fluorescent product was identified (390). Threonine and allothreonine TLC spots surements were also utilized (266, 602). develop a bright red fluorescence after treatment with Enzymes, Nucleotides, and Nucleic Acids. Advanninhydrin-collidine and 1% K O H in ethanol (871). Alatages of fluorometric assay of enzymes have been reviewed nine or other amino compounds were determined by fluo(309, 932). For the quantitation of hydrolytic enzymes rometry after treatment with o-phthalaldehyde and potasparticularly, a general method is to add a nonfluorescent sium borohydride (756); alanine was also assayed on a but fluorogenic substrate and measure the amount of fluomicro scale by a n enzymic method using glutamic-pyruvic rescence produced by the action of the enzyme under transaminase and lactic dehydrogenase (427).Another enstandardized conditions. Some examples were the assay of zymic-fluorometric procedure was utilized for homocarcholinesterase using 4-methylumbelliferone acetate as the nosine (1013). Urea in urine was determined by the 525substrate ( l o g ) ,of pancreatic lipase with 4-methylumbelnm fluorescence after treatment with the specific reagent liferone laurate as substrate (965), of microbial esterase diacetyl monoxime (745), and sialic acid was assayed by (666) and soil lipase (667) with the corresponding butyrate heating with diluted perchloric acid a t 100" for 10 minutes ester as substrate, of @-glucosidasewith 4-methylumbellifand reading the fluorescence of the propanol extract at erone P-D-glucoside as substrate (1477, and of serum hexo490 nm (880). saminidase, in screening for Tay-Sachs disease, with 4Fluorescamine, which was discussed earlier as a n immethylumbelliferyl-@-D-N-acetylglucosaminide as subportant new reagent for primary amines, is valuable also strate (145, 769). Closely related substrates were used to for amino acids and peptides (927) and proteins (83, 853). determine potassium-dependent phosphatase (909), pheThe fluorescamine reaction has been used to achieve a nolsulfotransferase or 3'-phosphoadenosine 5'-phosphosulgreat increase in sensitivity for column chromatography fate (403), a-chymotrypsin and trypsin by a titration amino acid analyzers, down to the picomole level, through method (402),and 5'-nucleotide phosphodiesterase (541). use of a fluorescence detector in place of the colorimetric Another group of substrates consists of nonfluorescent ninhydrin detector (258, 854). Although proline and hyderivatives of naphthols or @-naphthylamine, the fluoresdroxyproline do not undergo the fluorescamine reaction cence of which is measured after enzymic hydrolysis. This because they are secondary amines, they can be included is illustrated by assay of aminopeptidases with L-leucylin the overall amino acid analysis by conversion to prifl-naphthylamide as substrate (330, 836, 931), of phosphamary amines through reaction with N-chlorosuccinimide tases with naphthyl phosphates (65, 223), of glutamylto produce oxidative decarboxylation, followed by hydroltranspeptidase with glutamyl-P-naphthylamide (620), of ysis of the resultant imines to primary amines (221, 976). cathepsin B-like activity in lyosomes with benzoylarginine The fluorescamine test has been applied for rapid detecfl-naphthylamide (169), and of hydrolases such as trypsin tion of trace amounts of uncoupled materials during synby a kinetic method (123). thesis of peptides on solid-phase resins (220).For a study In the case of enzymes which require as a coenzyme of the kinetics of solid-phase peptide synthesis, other some form of nicotinamide adenine dinucleotide (SAD), a workers used a fluorometric method based on DANSYL general procedure for assay is to measure the change in labeling (253). fluorescence intensity (Le., concentration) of the reduced Amino acids were quantitated a t the picomole level, by or oxidized form of NAD under specified conditions, fluorometry of their DANSYL derivatives (12, 946, 981). which results from the enzyme reaction. This method was The fluorescence of these derivatives in aqueous media utilized for the determination of glutamate-oxalacetate was greatly enhanced by the addition of cycloheptaamytransaminase (271); glutamate-pyruvate transaminase lose (458). DANSYL labeling was employed in a proceand hexose phosphate isomerase (375); transketolase dure for measuring the molecular weight of proteins (139); lactate dehydrogenase on a semisolid surface (1012), and 5-dibutylaminonaphthalene-1-sulfonylderiva(1023); glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in red cells (173, ,545);glutatives were proposed a s a less polar alternative to DANSYL thione reductase, glucose-6-phosphate dehydrogenase, and derivatives for fluorescence tagging of primary or secongalactose-1-phosphate uridyltransferase in deficiency dary amino groups, as in peptides or amino acids (803). screening ( 6 7 ) ;intestinal lactase, by coupling to the assay The peptide hormones glucagon and secretin showed of liberated galactose by galactose dehydrogenase (156); strong fluorescence after reaction with o-phthalaldehyde galactose-1-phosphate uridyltransferase in screening for (316, 318); the same reagent was used for visualization and determination of proteins in gel electrophoresis (975). galactosemia (268, 782); creatine kinase isoenzymes in A sensitive fluorescence reaction for amino acids is conserum (844); and enolase, pyruvate kinase, and lactate densation with pyridoxal, followed by either reduction dehydrogenase in single nerve cells (485). NAD fluorometwith borohydride (553) or chelation with zinc ion (558); ric techniques were used also for the automated analysis the latter was adapted to the automated assay of amino of glycolysis metabolites (796) and for the study of oxidaacids in protein hydrolyzates (560). Other reagents used in tive metabolism in rabbit muscle (130). fluorometric analyses of amino acids were fluorescein isoMany other fluorometric procedures were also used for thiocyanate (440), 7-chloro-4-nitrobenzo-2-oxa-1,3-diazoleenzyme assays. Proteolytic activity was measured with high sensitivity by reacting fluorescamine with the liber( B O ) , and o-diacetylbenzene in methanol as a spray for chromatogram spots of some rare amino acids (487). Imated amino acids (794);similarly, lyosomal peptidase was provements were made in the assay of protein by its quantitated by the fluorescence of the amino acids proquenching of the fluorescence of eosin Y (46). duced by reaction with o-phthalaldehyde and mercapAn interesting application of fluorescence measuretoethanol (894). Carboxypeptidase A was determined with ments, the study of macromolecular conformation, has the aid of DANSYL-labeled substrates (508). o-Phthalalbeen reviewed for proteins and peptides (99) and for biodehyde served as the fluorogenic reagent in the assay of logical membranes (729). Changes in the native fluoreshistaminase (461), carnosinase (627), and tryptophan-5cence of the protein, which are often due to changes of the hydroxylase (252). For tryptophan-2,3-dioxygenase,the immediate environment of tryptophanyl residues caused product was converted by additional coupled enzymic by folding, denaturation, etc., were studied in papers on: reactions to anthranilate, which was measured by fluohuman growth hormone (557); G- and F-actin (521);foldrometry (125). Aryl hydrocarbon hydroxylase was quanti508R
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tated by the increased fluorescence at 520 n m of benzo[alpyrene (480). In the assay of creatine kinase, the creatine formed was reacted with alkaline ninhydrin to give a fluorophore (484, 750). Cathepsin D was quantitated with high sensitivity by reacting the released tyrosine with 1nitroso-2-naphthol (594). Amino acid oxidase was measured by the decreased fluorescence of scopoletin (6-methoxy-7-hydroxycoumarin) in a coupled reaction (982); coupled reactions involving homovanillic acid were also used t o assay monoamine oxidase and diamine oxidase (839). Carbonic anhydrase bands on polyacrylamide gels were detected either by their native fluorescence at -70" (672) or by their strongly fluorescent complexes with DANSYL sulfonamide (365). In the nucleotide field, there was a significant advance: the synthesis of intensely fluorescent derivatives by reaction with chloroacetaldehyde in aqueous solution under mild conditions, such as 1,N6-ethenoadenosinefrom adenosine (53). This reaction has been applied to the fluorometric assay (excitation 312 n m , emission 420 n m ) of adenine and its derivatives (42) and to the detection of TLC spots of adenine-containing compounds (522).Fluorescent N-etheno derivatives have been prepared and studied for adenosine monophosphate (AMP) (678, 799), adenosine triphosphate (ATP), with retention of biochemical function (798), a series of adenosine phosphates (800, l o l l ) , cytidine and NAD (54, 800), polyadenylic acid (520, 856) and flavin adenine dinucleotide (FAD) (339). Adenine and its derivatives were also determined by the strong fluorescence they give when reacted with glyoxal hydrate trimer (1014).Reviews were published on the use of firefly luciferin-luciferase for luminescence assay of cyclic adenosine monophosphate (193, 409) and of AMP, ADP, and ATP in mixtures (502). Fluorometry of NAD and related coenzymes was used: in a paper chromatography method for oxidized pyridine nucleotides (770),in the simultaneous determination of ATP and reduced NADP (562), in automated analyses for micromolar quantities of ATP, glucose, and lactic acid (519),and in assays for succinyl- and propionyl-coenzyme A (838). A rapid micromethod for flavin mononucleotide and FAD was based on their differences in fluorescence a s a function of p H (209). The phosphorescence and fluorescence of 16 dinucleotide isomers were reported (477); the effects of p H on the phosphorescence of cytidine, cytosine, and its nucleotides a t 77 "K were studied ( 2 ) ; and the temperature dependence of the weak 550-nm emission of 4-thiouridine was measured (811). NAD analogs were prepared in which adenine is replaced by fluorescent purines such as 7-deazapurine, and which retain coenzyme activity and are highly fluorescent in both oxidized and reduced form (969, 970). For the microdetermination of nucleic acid, extensive use was made of a specific method in which the fluorescence intensity of ethidium bromide (2,7-diamino-l0ethyl-9-phenylphenanthridium bromide) is greatly enhanced upon binding to nucleic acids (113, 421). This technique was applied to the determination of deoxyribonucleic acid (DNA) in human granulocytes (74, 75), of DNA and RNA in tissue homogenates (428, 717), of DNA in bacteria (180), and of DNA interstrand cross-links (112) and bihelicity (618). Reagents other than ethidium bromide which have been used for luminescence studies or assays of nucleic acids are acridine orange (237, 1003, 1004), terbium ions, which complex and fluoresce by accepting excitation energy from guanosine residues (234), malonaldehyde (743),silver ion which was employed for a phosphorimetric determination of submicrogram quantities of DNA (730, 825), berberine sulfate (313),coriphosphin (9301, and auramine-0 in a Feulgen reaction (914). Fluorescence assays of DKA were found valuable in the diagnosis of cancer (80, 850),because more DNA than usual is present in malignant cells. Steroids and Hormones. Fluorometric assay of steroids generally makes use of the fluorescence developed by treatment with strong acids, sometimes in the presence of other reagents; a review was published on this subject (877). T h e 11-hydroxycorticosteroids cortisol and corticosterone were determined in blood or urine using a 7:3 HzS04-ethanol reagent or variations thereof (443, 632, 728, 872) or with methanol in place of ethanol (735). Interfering fluorogens were separated by methylene chlo-
ride/water partition (277), T L C on silica gel (776), or phase-separating filter paper (599).T h e specificity of cortisol fluorometry was investigated and improved (465, 598, 697), and interference by 21-deoxycortisol was found (142). Other aspects of the procedure were discussed (530, 937). Testosterone and epitestosterone were similarly assayed by fluorometry (excitation 600 nm, emission 620 n m ) after paper chromatography separations (196, 197). These same androgens were determined by TLC on alumina, with the 440-nm fluorescence of the spots being developed simply by heating 20 min a t 180" (199). In a spectrofluorometric method for methyltestosterone, the fluorescence was developed by HC1 and stabilized by ascorbic acid (18).Fluorescence spectra of several ketoandrostanes were studied as a function of the position of the keto group, and no phosphorescence was observed in these compounds (980). Testosterone urinary levels in hypogonadic and normal men were compared by a fluorometric procedure (488). Estrogen analyses by fluorometry and other methods have been reviewed in a Japanese article with 98 references (647). A useful fluorometric determination of estrone, estradiol, and estriol in urine or blood is the Ittrich -Kober procedure, in which sulfuric acid treatment is followed by extraction into a 2% solution of p-nitrophenol in tetrachloroethane or similar solvent (6, 272, 517, 663); preliminary hydrolysis of conjugates could be accomplished by glucuronidase ( 5 ) or boiling water (579).Automated procedures for urinary estrogens were described (149, 870). For progesterone, the optimum fluorescence intensity was obtained with 6:4 HzS04-ethanol a t 60" for 10 min (178). For mestranol, a 1:l HzS04-methanol reagent was used in the fluorometric assay (574). The Liebermann-Burchard reagent, which is trichloroethane-H$304 -acetic anhydride, served for the determination of ethinylestradiol by fluorometry (399).Other fluorescence methods were described for norgestrel (831) and estradiol valerate (397). Cholesterol was determined by its fluorescence on silica gel TLC plates containing ammonium sulfate, after prolonged heating a t 150" (610). Ecdysterone in nanogram amounts was assayed fluorimetrically after treatment under specified conditions with sulfuric acid (265). For spironolactone and its metabolites in biological fluids, 62% HzS04 was chosen as the fluorogenic reagent (767). Fluorometric procedures for conjugated bile acids in biological fluids included a preliminary splitting of the conjugates with alkali, TLC separations, and development of the fluorescence by spraying with HzS04-methanol and heating (102, 218, 260). For total bile salts in bile, the fluorescence was developed by HzSO4 treatment in solution (621). Prostaglandins were determined by a fluorometric NAD method, using prostaglandin dehydrogenase (653). Pharmaceuticals. Recent developments in analytical methods for pharmaceuticals, including fluorometry, have been listed in a n extensive review with 2670 references (144). An automated method for morphine in urine was based on its oxidation to the fluorescent dimer of pseudomorphine (774). Morphine, codeine, and heroin form strongly fluorescent complexes with cuprous halides on filter paper (974). Fluorometric procedures for detection of morphine and other illicit drugs were reviewed (670), and a combined fluorescence-gas chromatography met hod gave improved reliability in mass screening for such drugs as morphine, cocaine, methadone, and metamphetamine (775). Hashish smoking was detected by the presence in saliva of tetrahydrocannabinol, identified fluorometrically as its DANSYL derivative (247). T h e DANSYL derivatives of nine different cannabinoids were separated by TLC; the sensitivity of detection was less than a nanogram (235). A fluorometric assay for cannabinoids in blood or urine was based on condensation with a polycarboxylic acid and acid catalyst t o give a highly fluorescent compound (107). For amphetamine in urine, a n automated procedure utilized condensation with formaldehyde and acetylacetone to give a n intensely fluorescent dihydrolutidine derivative, but some other amines interfered (347).The fluorescence and phosphorescence spectra of amphetamine and six other phenylethylamines, ten barbiturates, and some model compounds were examined, and the results
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used for assay of pharmaceuticals (605). Oxybarbiturates in blood were determined by their luminescence a t 77 "K in 0.1M NaOH solution, with a sensitivity in the microgram range (264). Fluorescence and phosphorescence spectra of 48 psychotropic derivatives of thiophene, benzthiophene, and benzothienopyridazine were reported (899). Oxazepam was assayed by the 475-nm fluorescence resulting from treatment with phosphoric acid a t go", and the structure of the fluorophore was elucidated (117). For flurazepam and its metabolites in blood and urine, the fluorometric procedure was based on hydrolysis to the benzophenones followed by K&O3 cyclization to the 9acridanones (172). In the blood of schizophrenic patients, 11 chlorpromazine metabolites were quantitated as their DANSYL derivatives (439); the effects of pH, temperature, and solvent on the fluorescence of four phenothiazine derivatives were studied (671). A sensitive fluorometric method for protryptyline was found free of interference by other common psychiatric drugs (615).Amitryptyline and tetrabutylammonium ion were measured fluorometrically after ion pair extraction with anthracene-2-sulfonate (503).Ion pair extraction with fluorescent tetrabromofluorescein derivatives was also used to determine long chain secondary or tertiary amines such as amitryptyline or chlorpromazine (379) and tetraethylammonium bromide (380). Fluorometric and other analytical methods for lysergic acid diethylamide were discussed (857), and the fluorescence and phosphorescence parameters a t 25" and -196" were obtained for this psychoactive drug and 8 related ergot alkaloids (89).In TLC analyses for dihydroergotamine (87) and ergometrine and ergotamine, the fluorescence intensity of the spots was increased and stabilized by impregnation with 1:9 mineral oil-ether (248). The fluorometric assay of reserpine and related rauwolfia alkaloids by treatment with nitrous acid was semiautomated (412); an automated procedure for these same compounds used vanadium pentoxide as a reagent (935).Addition of thorium ion to a n ethanol solution of reserpine greatly increased the latter's fluorescence a t 500 nm (419). Direct fluorometry of TLC spots served well for the determination of reserpine, digoxin, and many other active substances in extracts of medicinal plants (328), and various techniques for such determinations of alkaloids were discussed (578). Improvements such as extraction separations were reported in quinidine and quinine analyses by their native fluorescence in dilute acid (34, 995). Cardiotonic glucosides, flavonoids, and coumarins were assayed by the fluorescence of their complexes (290),and 4-methylesculetin was suggested as a possible p H indicator on the basis of a study of p H effects on its fluorescence (280). Tetracyclines were determined by fluorometry of their chelates with magnesium (528), calcium (996),aluminum (134) and beryllium (22, 23). The role of molecular, structure was discussed in the formation of fluorescent chelates of several tetracyclines with Mg, Ca, Zn, Mn, and Cu (570). Prolonged heating of tetracycline in 0.1N K O H gave a fluorescent product which was used for quantitation (779). Other papers described the fluorometric assay of oxytetracycline in premixes (438), the TLC separation and fluorometry of tetracycline, anhydrotetracycline, and their epimers (942),and the fluorometric determination of demethylchlortetracycline and tetracycline in rat bone (344). Antibiotics containing amino sugars, such as kanamycin and polyoxin, were assayed by reaction with pyridoxal and zinc ion in pyridine-methanol solution to yield highly fluorescent zinc chelates of N-pyridoxylidenes (559). Griseofulvin was determined in mixtures with dechlorogriseofulvin, by means of time-resolved phosphorimetry (587), and a detailed study of its luminescence characteristics was reported (6411. Luminescence spectra of actinomycin antibiotics in the fungal cultures were found to differ from those in organic solvents (707). Polyene antibiotics such as filipin were found to undergo changes in fluorescence intensity and polarization upon binding to cholesterol, and were suggested as a probe for lecithin-sterol interactions (70, 183). Quenching fluorometry with 9-chloroacridine was employed for the determination of sulfonamides and local anesthetics which are primary amines (862). Phosphorescence and fluorescence 510R
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spectra were compared for a number of sulfonamide hypoglycemic agents (725). The muscle relaxant dantrolene and two of its metabolites were measured fluorometrically, after extraction and chromatography separations (367). Practolol, a p-adrenergic antagonist, after deacetylation forms a highly fluorescent conjugate with nitrosonaphthol, which is suitable for quantitation in blood and urine samples (78). Pancuronium bromide, a quaternary ammonium neuromuscular blocking agent, was assayed by the fluorescence of its complex with rose bengal dye after extraction into a n organic phase (446). For diphenylhydantoin, alkaline permanganate oxidation gave benzophenone, which was extracted into heptane and reacted with HzS04 to give a fluorescent product suitable for quantitation ( 17 4 ) . The native fluorescence of N-arylanthranilic acid analgesics such as flufenamic acid was found more useful for their fluorometric determination (596) than that obtained by derivatization procedures (168). Fluorescence and phosphorescence calibration curves were found to be linear for several antihistamines having 2-aminopyridine structures, the effect of p H was studied, and a fluorescence analysis of sleep tablets for their methapyrilene content was performed (994). Tripelennamine in anticold preparations was determined fluorometrically by heating with cyanogen bromide (661). Daunomycin and adriamycin in ascites cells and tissues were released from bound form by treatment with silver, and then assayed by fluorescence (795). For rifampicin quantitation, oxidation by H202 gave a product that fluoresced a t 480 nm in alkaline solution when excited a t 370 nm (224). Other papers report the fluorometric determination of isonicotinic acid hydrazide (529, 648); of pyrimethamine and related diaminopyrimidines on TLC plates, with enhancement of the fluorescence after spraying with NHIHSOI (833);of nifedipine by Tic13 reduction of the nitro group to amino, oxidation of the dihydropyridine ring to pyridine by ultraviolet irradiation, and condensation of the amino derivative with o-phthalaldehyde (784); of isoquinolines of pharmacological interest (393);of dequalinium compounds in urine, after TLC separation of interfering substances (167); of chlorphenesin carbamate and mephenesin (267); of mycophenolic acid and its glucuronide metabolite in blood (86); of thymoxamine in blood (32);and of thonzylamine by periodate oxidation to p-anisaldehyde, which was then condensed with o-aminothiophenol to produce a benzothiazole which fluoresced in strong acid solution (634). Agricultural Chemicals and Products. Recent extensive reviews cover fluorometric and other methods for the analysis of food (1009) and pesticide residues (904).Aflatoxins and other poisonous metabolites of molds continue to be determined by direct fluorometry of their TLC spots, in rice and wheat (718), in products containing alcohol (616),and in animal tissues (754), with provision for removing interferences by a silica gel column (732) or by two-dimensional chromatography if necessary. For the fluorometric determination of aflatoxin B1 in aqueous solution, treatment with 0.1N H z S 0 4 resulted in increased intensity and decreased photosensitivity (797).Low temperature luminescence measurements permitted the detection of nanogram amounts of aflatoxins (381);high sensitivity was also achieved by use of micro columns layered with Florisil (952).The absorption and fluorescence spectra of aflatoxins Bza and Gza were determined in different solvents (561).Much of the observed fading of fluorescent TLC spots of aflatoxins was found to be due to the cooling effluent air from the xenon lamp, and could be prevented by covering the plate with another glass plate (643).Aflatoxin M fluorescence standards were more stable when stored in chloroform solution in borosilicate glass test tubes than under other conditions (726).The fluorescence. phosphorescence, and absorption parameters of ochratoxin A were reported (642). Organophosphorus pesticides such as azinphosmethyl (245) are often determined by TLC methods, one approach being to develop the fluorescence by spraying with strong acid or base before heating (103). Other spray reagents used were flavones such as robinetin (569, 1,2-dichloro-4,5-dicyanobenzoquinone in benzene for malathion and similar compounds (239), and bromination followed
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by a mixture of manganous chloride and salicylaldehyde 2-quinolylhydrazone for organothiophosphorus pesticides such as parathion (244). Also reported were fluorometric pesticide assays based on cholinesterase interactions (356), on lipase splitting of fluorescein esters ( 7 ) , and on the transient intermediates formed in the reaction with sodium perborate and indole (763). The fluorescence and Raman spectra of some major pesticides, excited by a laser, were measured (955). For carbamate pesticides such as Sevin, fluorometric analyses made use of the DANSYL derivatives (511), with attention to their stabilization of intensity (513), TLC separation procedure (514), and in situ quantitation technique (242). Matacil and Zectran were determined by this method (241).A variation of the procedure is to first hydrolyze the carbamate or urea pesticides with hot 1M NaOH, and then prepare the DANSYL derivatives of the amine moieties for fluorometry (243). A rapid sensitive fluorometric method for Sevin was reported (476).Benomyl and some other pesticides were measured by means of their native fluorescence (566, 674). Phosphorescence spectra and other properties were reported for 13 chlorinated dibenzo-p-dioxins (705). The spatial localization of herbicides on leaf surfaces was observed with a scanning electronmicroscope equipped to detect cathodoluminescence (659). A simplified and improved method was developed for the fluorometric determination of ethoxyquin in feeds (999). Fluorescence and absorption spectra were observed for nalidixic acid and other l,%naphthyridines (604). In the assay of decoquinate in chicken tissues by its own fluorescence, a collaborative study showed good results (868). Dulcin was determined by its fluorescence after reaction with sodium nitrite (926);dulcin and saccharin were detected by the fluorescence of their spots after naphthylamine-cupric acetate spray or treatment with Auramine dye (582).Turmeric in foods was detected by the fluorescence of its principal component, curcumin, a t 520 n m in water-saturated butanol (426). Free flavines in plant tissues were investigated with aid of their fl-uorescence (912), and the luminescence of the neutral and cationic forms of flavines a t -196" were studied (534). Other papers reported the fluorescence of apple juice and concentrates, as dependent on the variety (940); a quantitative assay of the laxative sennosides as their fluorescent hydrazine derivatives (522);the phosphorescence characteristics and detection limits a t 77 OK for 17 plant growth hormones such as 2,4-D and naphthaleneacetic acid (772); fluorescence characteristics of fulvic acid and various humic compounds (533) and of natural and synthetic hymatomelanic acids (263); and the determination of fluorescent whiteners on fibers (8, 486) and as extracted from wrapping paper by foods (490). Fluorescence intensity was used as a n indicator of the freshness of meat (907). Immunofluorescence. This powerful technique permits the detection and quantitation of many molecular species used as antigens, by their immunochemical reaction with fluorescence-labeled specific antibodies followed by examination by fluorescence microscopy. Recent reviews cover current developments (215), standardization and quantitation (960), localization of bacterial antigens (967), and procedures for labeling serum proteins with fluorescein isothiocyanate (406). Applications of this technique to the diagnosis or other aspects of disease were reported for typhoid (1001), cholera (171), salmonellosis (387, 778), leptospirosis (179, 363), malaria (184),syphilis (348),rabies (913),tularemia (934), rickettsial infections (50), lupus erythematosus (922) including the inhibitory effect of heparin (1017, 1018), influenza and related viruses (809), infectious mononucleosis (191),and the detection of antibodies to milk proteins and gliadin in patients with celiac disease (106). Some other uses were the localization of water-soluble proteins in the wheat endosperm (51), the detection in human tissues of hormones (606) and collagen (759),the localization of alkaline phosphatase in bovine intestine (881), the detection of type E botulinal toxin in cultures ( I ) , the demonstration of cathepsin D in the joints of rabbits with experimental arthritis (712), detection of rheumatoid factors (2051, the detection of cytotoxic antibodies (201), the study of hemolytic streptococci antigens (761, 963), the
detection of tumor cells (262, 617), the binding of lectin to the surface of mammalian cells (483), and showing the exposure of cattle to infection by trypanosomes (39) and adenoviruses (875). Another group of papers dealt with refinements in immunofluorescence techniques, such as better methods for the preparation (218, 407) and purification (628) of fluorescein isothiocyanate or its conjugates; inhibition of nonspecific fixation of fluorescent globulins ( I 14); the separation of optimally labeled fragments of immunoglobulin G (236); a spectrophotometric determination of fluorescein/ protein ratios in conjugates (105); the use of 488-nm laser excitation for the fluorescence (961); a simple and inexpensive method for quantitation of immunofluorescence (340); a n automatic recording device (466);the effect of solubility changes after fluorescein- and DANSYL-labeling of immunoglobulins (36); standardization with artificial sections of selected antigenicity (95);a new label for primary amino groups which produces intensely fluorescent N-substituted 3,5-diphenyl-5-hydroxy-2-pyrrolin4-ones (977); and the applications of fluorescence polarization measurements to immunoassay (157). A related field is non-immunologic fluorescence microscopy, which has been the subject of a long review in Russian (597) and a shorter one in English (785).One specific aspect of interest is the detection or quantitation of DNA and chromatin material by fluorescence staining with acridine orange (164), quinacrine mustard (719, 1019), auramine (493, 983), a benzimidazole derivative (355), berberine sulfate (919), or pararosaniline Feulgen (249). A second area of interest is the cytofluorometric demonstration of catechol amines and other biogenic amines by exposure to formaldehyde vapor (182, 658, 837), which has been the subject of a review with 82 references (251); the reaction mechanism and nature of the fluorescent products were established (72). Subsequent treatment with HC1 intensifies and shifts the fluorescence, enabling differential identification of noradrenaline and dopamine (71, 31 7, 623). Tryptophan derivatives also were identified by their HCHO-induced fluorophores (208, 322, 323, 874). Reagents other than HCHO which were found to give similar fluorescence histochemical reactions with biogenic amines included glyoxylic acid vapor (73), o-phthalaldehyde (150), paraformaldehyde-mineral oil (771), and many other carbonyl compounds (44, 755). Some other applications of fluorescence microscopy were the localization of vitamin A in the pregnant rat (255), the detection of carbohydrates in animal tissues by staining with fluorescein-labeled concanavalin A (864), the distinguishing of elastin from collagen by their different affinities for phosphomolybdic acid (723), the determination of histones and total proteins in cells by staining with sulfaflavine (Lila), and the demonstration of callose in plant parts such as sieve tubes, by staining with aniline blue (680, 731). Miscellaneous. Fluorescence reagents reported for detection and quantitation of sulfhydryl groups were N-(9acridiny1)maleimide (638), N-(4-anilino-l-naphthyl)m aleim ide ( 4 2 2 ) , N - ( p -(2-benzimidazoly1)phenyl)maleimide (806), and N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid and its 1,8-isomer (384). Fluorometric assays of porphyrins in urine made use of TLC separation as their methyl esters (181, 695) and of their fluorescence when adsorbed by talc in acid medium (66). The fluorometric microdetermination of protoporphyrin in blood was found valuable for detection of subclinical lead poisoning in children ( 777). Related papers discussed hematoporphyrin as a diagnostic tool for malignancy by its induction of fluorescence in the tumor (773), the phosphorescence and delayed fluorescence of zinc porphyrins in microorganisms (832),the heavy atom effect in the luminescence of tetrahalo derivatives of tetraphenylporphine and their Mg and Zn complexes (843),the phosphorescence of metalloporphyrins in liquid solution (920), oxidized and reduced states of porphyrins in intact cells of Candida yeast, as shown by luminescence (569), and the phosphorescence properties of hemocyanin (810). The fluorescence of chlorophyll, in relation to photosynthesis, continues as an active field and the subject of reviews (287, 509). Fluorometric determination of the ratio of concentration of chlorophyll a to chlorophyll b was
A N A L Y T I C A L C H E M I S T R Y , V O L . 46, N O . 5, A P R I L 1974
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more sensitive in EtOH solution a t 77 "K, using excitation a t 478 n m and measurement a t 658 nm and 678 nm (77). In the fluorometric assay of chlorophylls a, b, and c in mixtures with pheophytins a, b, and c, it was necessary to use six simultaneous equations for the measurements at three different wavelengths, before and after acidification (542). Procedures were described for the fluorometric estimation of any one or more of the 8 pigments consisting of chlorophylls a and b, chlorophyllides a and b, pheophytins a and b, and pheophorbides a and b (988). The effect of p H on fluorescence and absorption spectra of pheophytin was studied (936), and quasiline spectra of chlorophylls were observed a t liquid helium temperature (41). For bacteriochlorophylls in vivo and in vitro, the temperature dependence of absorption and fluorescence spectra was measured (274). Bacterial bioluminescence was studied as a function of the oxidation of reduced flavin mononucleotide (516). An assay of the potency of anesthetic agents such as halothane was based on changes in the luminescence of Photobacterium species (986, 987). The ultraweak luminescence of seedlings was used as a n indicator of metabolic disturbance after treatment with salt solutions (432) or substances such as glycerol or urea (10). The hydroxylating action of liver microsomes was accompanied by the emission of luminescence in the presence of luminol(162). The dye intermediate Koch's acid, which is 8-amino-
LITERATURE CITED Aalvik, B.: Sakaguchi, G . ; Riemann, H.. Appl. Microbioi. 1973, 2 5 ( 1 ) . 153. Aaron. J J : Spann, W. J.; Winefordner, J D , Talanta 1973, 2 0 ( 9 ) , 855. Aaron, J J . ; Winefordner, J . D . . Anal. Chem. 1972, 44(13), 2122. Aaron, J. J . ; Winefordner, J. D , Talanta 1972. 1 9 ( 1 ) , 21. Adessi, G : Jayle, M . F . , Ann. Biol. Clin. (Paris) 1972, 30(3). 219; Chem. Abstr. 1972. 77. 123546e. Adessi. G.: Jayle, M . F . , ibid.. 3 0 ( 2 ) . 127: Chem. Abstr. 1972, 77, 45078n Adonai Martin, E., Can. J . Pharm. Sci. 1972. 7 ( 1 ) . 21 Aehwal. W B.: Narkar, A. K , Textilveredlung 1972, 7 ( 1 ) . 19; Chem. Abstr. 1972. 76, 1146341 Afghan, B K . : Goulden. P. D ; Ryan, J F . , Water Res. 1972, 6 ( 1 2 ) , 1475 Agaverdiev, A Sh.; Tarusov, B N , Biofizika 1973, l S ( l ) , 94. Ahmed, M . : Davies, A. K . : Phillips. G. 0 : Richards, J T., J. Chem. Soc.. Perkin Trans 2 1973, ( 10) , 1386 Airhart, J : Sibiga, S.: Saders, H . ; Khairallah, E A , Anal. Biochem. 1973, 5 3 ( 1 ) . 132 Akaishi, J . ; Yabe. A , Jap. At. Energy Res l n s t . , (Rep.) 1969, JAERI-13622, 21 pp: Chem. Absfr. 1972, 76. 10058h. Akhmedli. M . K . ; Efendiev. D. A.; Ruvinova, F I , Azerb. Khim. Zh. 1972. ( 3 ) . 146. Chem. Abstr. 1973, 79, 121575f Akimov. I A,. Opt. Spektrosk 1972, 3 2 ( 2 ) . 317; Chem. Abstr. 1972, 76, 147018q Alferov, G N ; Donin. V I ; Chebotaev. V P.: Yurshin, B. Ya.. Zh. Prikl. Spektrosk 1973, 1 8 ( 2 ) . 316; Chem Abstr. 1972. 78, 11 7 2 2 0 ~ . Alikov. Kh K : Aliev, R Z . ;Alykov, N M . : Gorbanov, R . F . Tr. Konf. Moiodykh Nauch. Sotrudnikov Astrakhan. Med. inst.. 2nd 1969 (Pub. 1970). 97; Chem. Abstr. 1973, 78. 64648k Alkalay. D . : Khemani, L.; Bartlett. M . F , J . Pharm. Sci. 1972, 61(11), 1746 Almqren, M , Mol. Photochem 1972. 412); 213. Al'shits, E . I ; Godyaev, E. D . ; Personov. R . I . . lzv. Akad. Nauk SSSR. Ser. Fiz. 1972, 3 6 ( 5 ) , 1117: Chem. Abslr. 1972. 77. 81 745m Alykov. N M ; Aliev, R. 2 . . Tr. Konf. Molodykh Nauch. Sotrudnikov Astrakhan. Med. l n s t . 2nd 1969 (Pub 1970i, 91; Chem Abstr 1973, 78 64646h (22) Alykova T V Antibiot,ki /Moscow)
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1,3,6-naphthalenetrisulfonicacid, was determined by its emission a t 520 nm, excited a t 360 nm (591). In phloxine solutions in ethanol, the fluorescence intensity was measured as a function of p H and concentration (279). The phosphorescence of trypaflavine adsorbed on cellulose triacetate thin films was found to be insensitive t o oxygen, in contrast to the well known oxygen sensitivity of the same dye adsorbed on silica gel (835). The luminescence spectra of cyanine dyes in the solid state were reported (15), and the fluorescence and absorption spectra of different ionic forms of proflavine and acridine orange were measured (962). Azotols in organic pigments were determined by fluorometry (312). The concentration of various types of surfactants, such as laurylpyridium sulfate and triethanolamine lauryl sulfate, in solutions was measured by their native fluorescence (424, 425). Dansylglycine was used as a fluorescent probe for micellar behavior of cationic detergents (161). Fluorometry was used for the examination of polymer films (592) and for the determination of low molecular weight fractions in polystyrene, using Rhodamine 6G (948). Various types of strongly acidic organic anions, including sulfated polysaccharides, were found to give fluorescence with Pinakryptol Yellow on cellulose layers (630). The literature of fluorometric analysis has grown so large that it is difficult t o do justice to this subject in a single review.
1972. 1 7 ( 4 ) . 353; Chem. Abstr. 1972, 77, 28708a. Alykova, T V ; Cherkesov, A I . ; Alykov. N M . . Izv. Vyssh. Ucheb. Zaved. Khim. Khim Tekhnol. 1972, 1 5 ( 7 ) , 1107; Chem. Abstr. 1972, 77, 105657n. Ambrose, J. A,, Ann. N . Y . Acad. S o . 1972, 196(Art. 5 ) . 295. Ando. I ; Horiuchi, Y ; Miura, A,; Harumoto, T., Arerugi 1973, 22(6). 435: Chem Abstr. 1973, 79, 1 3 4 0 9 0 ~ . Andrews. T M . ; Goldthorp, R.; Watts, R . W. E., Clin. Chim Acta 1973, 4 3 ( 3 ) , 379 Anikina, L. I ; Balashov, Yu. A , ; Vul'fson, E K ; Karyakin, A. V , Zh. Anal. Khim. 1972, 2 7 ( 4 ) , 787; Chem. Abstr. 1972, 77, 56047c Anikina. L . i ; Dobrolyubskaya, T. S . ; Karyakin. A. V.; Le Viet Binh, Khim. Svoistva Soedin. Redkozemel. Elem., Dokl. Vses. Soveshch. Fiz.-Khim. Primen. Redkozemei. Elem., I k h Soedin. Splavov. 6th 1969, 98; Chem. Abstr. 1973, 79, 121 557b Antipenko, B M . ; Batyaev. I . M . ; Lyubimov. E. I., Opt. Spektrosk. 1972, 3 3 ( 5 ) . 938; Chem. Abstr. 1973, 78, 77669t. Anton. A H . : Sayre, D. F . , J. Pharmacoi. Exp. Ther. 1971, 179(2), 207. Antonov. A. V : Melamed. Sh. G , Anal. Khim Redk. Metal. Poluprov. Mater. 1970, 149; Chem. Abstr. 1972, 76, 94254e. Arbab. A. G ; Turner, P., J. Pharm. Pharmacoi. 1971, 2 3 ( 9 ) , 719. Ardashev, A A ; Golikov. P. P., Biol. Med. lssled. Dal'nem Vostoke 1971, 95: Chem. Abstr. 1973, 78,40022a. Armand, J.; Badinand, A,. Ann. Biol. Ciin. ( P a r i s ) 1972, 3 0 ( 6 ) , 599; Chem. Absfr. 1973, 78, 7 9 5 2 9 ~ . Arnold, D. R . : Birtwell, R J , J. Amer. Chem. SOC. 1973, 9 5 ( 1 4 ) , 4599. Arnold, W ; Von Mayersbach, H.. J. Histochem Cytochem. 1972. 20( 1 2 ) , 975 Arsen'ev. V. V.: Gavanin, V. A.; Pashchenko, V 2 . ; Protasov, S. P ; Rubin, L ; Rubin. A. B , Zh. Prikl. Spektrosk. 1973, 1 8 ( 6 ) . 1093: Chem. Abstr. 1973, 79 59900c Artemova, L K ; Bolotnikova. T N . , Urai. Konf Spektrosk.. 7th 1971, No. 2, 137: Chem. Abstr. 1973, 78, 57175a Ashkar, T., Ochilo, M.. Bull. W . H . O . 1972. 4 7 ( 6 ) . 787. Astaf'eva, I N ; Shcherbov, D. P.; Plotnikova, R N , Issled. Obi. Khim. Fiz. Metod Anai. Miner. Syr'ya 1971, 30; Chem Abstr 1973, 78 66554a Avarmaa R Rebane K Eesti NSV
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