(1406) Zail, S. S.,Joubert, S. hl., Brif. J. Haematoi. 14, 57 (1968). 11407) Zak. B.. Welsh. B. A., Weiner, ‘ L. $I., J.’Chromafogr.34, 275 (1968). (1408) Zaki, 0. A., Khogali, A., Rabie, F., Abdel-Wahab, 11. G., Hassan, Y. hf., J . Trop. i l l e d . Hyg. 72, 75 (1969). (1409) Zakrzewska, A., filed. Dosw. Mikrobiol. 20,99 (1968). 11410) Zamfir. J.. Szabo, hal., Rev. Roum. Chim. 13,219 (1968). (1411) Zamiri, I., hlason, J., A‘ature 217, 258 (1968).
(1412) Zegers, B. J. M., Poen, H., Stoop, J. W., Ballieux, R. E., C h . Chim. Acta 22, 399 (1968). (1413) Ziegler, hl., Lippman, H. G., Acta Biol. M e d . Ger. 21, 733 (1968). (1414) Zoria, V. G., Klin. Khir. 1967, 43. (1415) Zubaidov, U., Dokl. Akad. Nauk Tadzh. SSR 11, 57 (1968); CA 69, 65101~. (1416) Zubareva, L. A,, Solomonova, 0. N., Kuznetsov, N. I., Kyazimov, S. B., Sel’skokhoz. Biol. 4, 89 (1969).
(1417) Zwaal, R. F. A., Van Deenen, L. L. M., Biochim. Biophys. Acta 163,44 (1968). (1418) Zwaan, J., Anal. Biochem. 21, 155 (1967). (1419) Zwvaan, J., Ezp. Eye Res. 7, 461 (1968). (1420) Zwaan, J., J . Cell. Physiol. 72, 47 (1968). (1421) Zwaan, J., Maki, T. N., LVature 218, 476 (1968).
Fluorometric Analysis Charles E. White, University of Maryland, College Park, Washington, D. C. 2 0 2 0 4
T
is the twelfth of a series of biennial reviews on Fluorometric Analysis and covers the period approximately from December 1967 to Ilecember 1969 (752). During this period several books and reviews have been published dealing with fluorescence analysis. Dr. C. A. Parker of England, a recognized international authority on fluorescence, has titled his book “Photoluminescence of Solutions with Applications to Photochemistry and Analytical Chemistry.” From both a theoretical and practical standpoint this book must be rated as one of t h e best published on the subject of fluorescence and phosphorescence (488). Another book from England, “Luminescence in Chemistry,” by Bowen is one of the Van Sostrand series in Physical Chemistry and is intended for students and research workers in the area of luminescenee (76). This book gives excellent background material but little direct analytical procedures. An enlarged and revised edition of Udenfriend’s book on “Fluorescence Assay in Biology and Xedicine” is a welcome updating of this classic (697). ‘(Fluorescence ilnalysisA Practical Approach” by White and Argauer contains specific laboratory outlines for fluorometric analysis in inorganic, organic, biological and clinical chemistry as well as background material (731). Volume I1 of Passwat’er’s “Guide to Fluorescence Literature” covering 1964-68 is advertised for publication early in 1970 (489a). A book on “Phosphorirnetry” by Zander translated from the German gives a clear exposition on the theoretical background of fluorescence and phosphorescence and discusses mainly the HIS REVIEW
1
Alfred Weissler is author of the Organic
and Biological Section.
Md. 2 0 7 4 2 , and Alfred Weissler,’ Food and Drug Administration,
phosphorescent properties of aromatic hydrocarbons with selected example$ of analysis (754). “Molecular Light” (Luminescence) has been published in Russian (704) and the “Theory of Luminescence’’ originally in Russian has been translated into English (641). Other books on Organic and Biological topics are listed under the appropriate sections below. The papers presented at two international conferences on “Luminescence” have been published in book form; one held at Budapest, 1966, is in two volumes (in English) with a total 2165 pages (664) and the other given at Loyola University, Chicago, 1968 (395). Both cover many aspects of luminescence and the papers appropriate to this review are listed under the subject matter headings. T h e following reviews and general articles are pertinent: a review of fluorometric analysis (609); a review of luminescence analysis given as part of the Symposium on 50 years of Soviet Analytical Chemistry (79); a chapter of Fluorometry and Phosphorimetry in a book on Instrumental Methods of Chemical Analysis (187); a nine page article on the theory of molecular luminescence by Kasha (526); a seven page article on the theory of fluorescence by Yoshide (761). The meaning of sensitivity in trace analysis is discussed (45). The shape of fluorescence and absorption bands have been calculated for several complex organic molecules and are found to agree with experimental resuIts (715). A method calculating the constants for temperature quenching of fluorescence of several organic molecules seems to have promise (694). One of the instrument companies (682) is publishing a series of review articles, on the fluorescence analysis of elements and compounds, which are free on request and will be referred t o under the appropriate head-
ings. Another of the instrument firms frequently has general articles in their bulletin which is also free on request and will be referred to under the various sections below (16). Quantum yields of fluorescent materials are of interest to the analytical chemist and quinine sulfate has always been a problem as a standard. X series of 8 samples of quinine sulfate or bisulfate were obtained from various suppliers and were examined for absorption and excitation spectra and quantum yields. Solid samples had only a 2.270 relative variation in quantum yields but a commercial liquid sample was 2070 low (203). The quantum yield of quinine sulfate has been shown to change with the sulfuric acid concentration; the values in O.lN, lN, and 3.6N acid were found to be 0.50, 0.54, and 0.60, respectively (245). This same article reports that the quantum yield of quinine sulfate varies with the excitation wave length; the yield was 0.48 with 313.1 nm and from 0.54 to 0.6 with 365.5 nm and 435.8 nm. Other workers believe that the quantum yield is independent of wavelength (218). A comparison of fluorescence quantum yield standards indicates that 9,lO-diphenylanthracene is 2.17 i 0.1 times greater than quinine sulfate (651). This suggests that the value of 0.55 for quinine sulfate is too large and should be 0.46, or 16% lower than the accepted value. The quant u m yields of tryptophan, tyrosine, isomeric tyrosines, and fluorophenylalanines have been reported (115). New data on the quantum efficiency and spectra of some important phosphors have been determined with a careful control of all parameters involved (677). The calometric, photometric, and fluorescent lifetime determinations and fluorescent yields have been determined for fluorescein and eleven halogenated derivatives (690). X vi-
ANALYTICAL CHEMISTRY, VOL. 42, NO. 5, APRIL 1970
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sua1 method has been suggested for determining quantum yields within 25% (27). APPARATUS
The American Instrument company’s new spectrofluorometer SPF125 is a relatively inexpensive double monochromator instrument designed for routine analysis (493). This instrument has a high pressure mercury vapor lamp as a light source, a manual scanning device, and provision for low temperature and constant temperature measurements. A new multipurpose luminescence spectrometer of the Farrand Optical Co. has been constructed to give corrected excitation and emission spectra (136). Another all-purpose spectrophotometer for fluorescence determinations equipped with two monochromators, interchangeable glass and quartz optics, and automatic recording is reported as sensitive to quinine sulfate a t 0.005 pg/ml (526). The design and performance details of a compensated versatile spectrofluorometer that plots directly quanta against wave number has been described (141). d recording fluorescence polarization photometer is designed to record polarization as a function of time or wavelength (151). The construction of apparatus, and principles of measurement of polarization of excitation, spectra have been reviewed (650). Another instrument designed to measure fluorescence or phosphorescence of amounts as low as 10 pl is called a variable temperature ultraviolet spectrograph (177). Several articles contain useful suggestions on increasing the utility of spectrofluorometers with film polarizers (669), a guide to slit width selection (492), and inexpensive flow through macro and microcuvets (668). There seems to be a growing interest in the measurement of very weak fluorescence emissions as witnessed in several publications. An image intensifier spectrograph has been designed to measure the weak emission from chemiluminescence or electroluminescence systems (456). A modification of the Spekker absorption spectrophotometer has been designed to measure 1 ng/ml of quinine sulfate (524). A photoelectric apparatus is designed to record weak fluorescence or phosphorescence spectra, absorption, excitation, and degree of polarization (42). A unique idea to extend measurements in the ultraviolet region places a 2 mm layer of liquid phosphor in front of the photomultiplier tube (530). The measurement of the fluorescence of materials on thin layer chromatographic plates is becoming increasingly important and most of the commercial spectrofluorometers have accessories for this purpose. The topic of direct measurements on thin-layer chroma58R
tographic plates has been discussed in a general article with 12 references (180). Many other papers on this subject are included in a book covering the papers of a Thin-Layer Chromatographic Symposium (313). Instruments for fluorescence measurements on paper strips are also in use (63). The use of fiber optics in analytical chemistry has been discussed in a general article (138) and is applied in thin-layer chromatographic scanning devices (256). An article on the distribution of energy in the xenon arc contains a great deal of helpful information in adjusting the arc. Curves and tables show the radiant flux a t various parts of the arc (491). The spectra of xenon and krypton arcs have been compared and the efficiency of the xenon was 55% compared to 35% for krypton as measured with a thermopile (457). An unusual device is used to obtain a strong radiant flux at 280 nm. This consists of a low pressure mercury vapor discharge lamp wrapped helically around a silica rod. The 254 nm radiation produced an incoherent radiation of 280 nm which exits from the polished end of the rod (162). This source is used in the photometric analysis of proteins. An improved collimator for extreme uv and x-rays consists of a simple form of multiple plate apertures where there is a random array of holes in identical plates (701). Cathode rays and x-rays are becoming popular excitation sources for the production of visible fluorescence in the analysis of the lanthanides and various minerals. A cathode ray source is furnished by Kuclide Corp. (468) in a n apparatus called a Luminoscope. This instrument consists of high voltage power supply and a vacuum stage with a n attached electron gun “engineered to provide maximum safety and operating ease.” An ion luminescent method of analysis has been used to determine the presence of All Xa, and Ca in minerals. The mineral was bombarded with K + ions and the analytical lines were photographed. The 396.1 nm and 422.7 nm lines were used for ill, 589 and 589.6 nm for N a , and 422.7 nm for Ca. The exposures were 1 min for Al, 2 min for Na, and one for Ca (427). A high frequency short wave generator has been designed for a fluorometer where the 254 nm line is enhanced and contains 85y0 of the energy emitted; the overall emission is from 230 to 605 nm (602). Laser excitation fluorescence combined with matrix isolation provides a highly specific analytical technique (614, 652). A miniature dark room 8 X 43/4 X 9 inches with 4 watt longwave uv tubes is advertised for porphyrin determinations (738). Data on two new standards for correction of fluorescence emission spectra have been published. The compound
ANALYTICAL CHEMISTRY, VOL. 42, NO. 5, APRIL 1970
2-aminopyridine in 0.1.4‘ HzS04 covers the range from 300 t o 400 nm (552, 674, and is available commercially. The other standard, p-(dimethylaminobenzylidene) hippuric acid is easily prepared, and in dioxane covers 400600 nm, and in D M F from 548-800 nm (176). Rhodamine B has been studied further as a photon flux standard from 250-600 nm and it is stated that from 250-310 nm and 490-590 nm the photons are all absorbed in less than 1 nm from the front surface (750). These differences have no effect on readings from the front surface. The use of digital computer techniques for correction of fluorescence spectra has been described in two papers (106, 1%). The methods of calibration of fluorescence apparatus have been reviewed (497). The source of a n irradiance standard lamp is given in reference 56 and another in ( 3 ) . A good discussion of light measurement from 200 to 1200 nm is given in a commercial brochure (299). A unique cell housing for the simultaneous measurement of transmittance, optical density, 90’ light scattering, and fluorescence is advertised for the Schafer/Pheonix-universal spectrophotometer and is said to be applicable to other commercial fluorometers (507). The use of a film actinometer to measure solar radiation from 280 to 410 nm may have application in evaluation of fluorometer excitation units; the quantum efficiency is independent of wavelength from 300 to 400 nm (611). I n a general article on fluorescence analysis, a comparison of the sensitivity of four commercial fluorometers (3 Soviet and one Zeiss) was reported for samples of quinine sulfate, fluorescein, Rhodamine B, and Rhodamine 6G. The sensitivity of several reagents for A1 and M g were also quoted (79). Studies on optimum conditions in photometric and fluorometric analysis contain several useful suggestions (363, 599,600). Articles on spectrophosphorimetry, microscopy, and fluorescence microscopy give a good discussion and a diagram of the apparatus (487). A vertical illuminator for fluorescence microscopy and a discussion of the excitation and emission are given (512). A description of the resolution with a single disk phosphorimeter (279) and suggestions for minimizing background in commercial cells provide helpful material (760). The purification of organic solvents for fluorescence spectrometry has always been a problem. The addition of paraffin wax to the solvent before distillation of light petroleum hydrocarbons, alcohols, tetrahydrofuran, and ethyl ether, produces an improved product (200). A filter-photomultiplier tube combination has been devised so that the recorded results of fluorescent colors will approach the human eye response (622).
INORGANIC
General. A review of fluorescence analysis of inorganic substances for t h e years 1962 to 1968 which covers 36 elements, CN- and N2Hl and lists 297 references has appeared in a USSR publication (594). A similar review covering a year in Japanese includes fluorescent indicators, reliable methods for 21 elements, and has 56 references (459). A general survey of the applications of luminescence analysis to inorganic substances contains practically no new material (87). A review article on the structure of 2'2'-dihydroxyazo compounds complexed with metal ions gives useful information on this type of reagent (564). Analytical applications of Schiff bases are reviewed with 66 references. The factors influencing the stability and color of the bases and chelates are discussed (316 ) . The relation between structure and analytical use of some basic dyes such as acridine and rhodamines are discussed and suggestions are made for improvement of the dyes as reagents (508). The compound 3,5-bis(dicarboxyaminomethyl)4,4'-dihydroxy stilbene is found to form fluorescent complexes with AI, Be, RIg, Sr, Ba, Zn, Cd, Yt, La, Ga, and Lu. The fluorescence and other properties of resorcylaldehydeacetylhydrazone complexes with Zn, Al, Sc, and Ga has been reported (703). The optimum p H conditions, excitation, and emission value have been determined (99). A review of the luminescence characteristics of the halogen salts of Bi, Sn, Sb, Te, Ga, Ge, As, Si, Cu, In, Au, and Ag a t -185 "C shows the application of this luminescence method of analysis (50). The color, absorption, and emission maxima and fluorescent lifetimes of the nitrates of Na, Cd, Ag, Hg(I), and Tl(1) and also the phosphorescent lifetimes of acetates and arsenates cf other metals have been presented (411). An excellent article on the fluorescent properties of 2,3-, 2,4-, and 2,5-dihydroxybenzylidene-o-aminophenols and their complexes with a number of diand trivalent elements shows a number of applications to analysis of metal ions. The 2,4- compound seems to produce the most intense fluorescence in the chelates and the complex with A1 in 70% DRIF at a p H value of 6.1-6.2 has the highest intensity (318). The solubility of the complexes of 8-quinolinol with Cu, Al, Fe(III), and Zr have been tested with 17 organic solvents, Aluminum and iron complexes can be separated from the others by extraction with butyl acetate; both chloroform and dichloroethane are also excellent solvents (757). The analytical applications of the flavonols as fluorometric and colorimetric reagents have been the subject of reviews by two authors: in one case 180 references are listed
(36%')and in the other 118 (328). The author of the latter article has also published a review on the analytical applications of the hydroxycoumarins (330). A study of the grade of water used for dilution in fluorometric analysis leads to the conclusion that ordinary distilled water and ordinary ion exchange water contain objectionable contaminants. Triple distilled water from glass stills is rated as excellent and also water from a commercial system that utilizes an activated carbon organic removal unit on a 1-micron filter and a resin deionizer is satisfactory (490). Aluminum. A summary of the fluorometric reagents for the determination of aluminum is one of a series of fluorescence reviews published by t h e Turner Instrument Co. (684). A comparative study of 7 reagents for A1 including morin, salicylidene-o-aminophenol, 5-chloro-2,2',4'trihydroxyazobenzene - 3 - sulfonic acid (lumogallion) , 8-quinolinol, quercetin, and Chromogen Black E . T . - 0 0 ((3.1. Mordanted Black 11) has been the subject of two communications (36, 37). Quantum yields for the chelates and excitation and emission curves are given. The product of the quantum yield and the molar extinction coefficient is suggested as a sensitivity index. Solvents used were methanol, ethanol, acetone, and DMF. The authors rated morin in 95% ethanol and salicylideneo-aminophenol in 67% ethanol as the most sensitive of the reagents studied. I n a later paper it has been shown that acetate and biphthalate buffer ions compete with salicylidene-o-aniinophenol for -41 ions and reduce the light emission. A buffer made with urotropin is recommended (32). Several new compounds made by coupling the diazonium salt of 1- amino-2-naphthol4-sulfonic acid with a number of coumarin derivatives have been tried as reagents for Al. The most successful was one formed with 4-methylumbelliferone which gives a pink fluorescence with A I (max. Em. 590 nm and Ex. 540 nm) a t a limiting concentration of 0.2 ppm and a p H of 4-5 ( 2 ) . A procedure for the analysis of cement uses fluorometric reagents for AI, Ca, and Mg. Salicylidene-o-aminophenol was used for All Calcein for calcium and lumo-magneson IREA, 5-(5-chloro-2hgdroxysulfophenyl) barbituric acid, for RIg (375). Lumogallion has been used to determine AI in sea water (460). These same authors previously reported on the optimum conditions for the reaction of this reagent for A1 and Ga and have listed the interferences (461). The fluorescent determination of A1 with 8-quinolinol has been used for soil samples (125) and the 2-methyl8-quinolinol complex with A1 in methanol has been fully characterized (574). A study of the fluorescent chelates of
A1 with flavonol in absolute alcohol are reported to be in 1:1, 2:1, and 6:l ratios of B1: flavonol (702). Beryllium and Boron. A new reagent has been developed t h a t is reported to be highly selective and sensitive for Be, La, and Lu. The name of the compound is 1-(dicarboxyaminomethyl)-2-hydroxy-3-naphthoic acid. It also forms fluorescent complexes with All Gal Hf, In, Mg, Se, Th, Y, and Zr. The Be, La, and Lu chelates have a 1 : l ratio of metal to ligand. The Be complex shows its maximum intensity a t pH 6.8 and its excitation fluorescence maxima are 360 and 450 nm. The linear analytical curve for Be is 0.091.8 pg. Interferences have also been determined (1O f ) . The inhibition by Be on the catalytic effect of calf intestinal alkaline phosphatase on the hydrolysis of 2-naphthyl phosphate which is monitored by measuring the fluorescence of the 2-naphthol has been reported as another method of analyses for Be. This method has been used to determine 18-90 ng of Be and 0.6-6 pg of Zn (695). An excellent review of the fluorescence reagents for Be has been compiled with 33 references (686). Improvements have been reported in the morin method for the determination of small amounts of Be in materials of complex composition (211, 51 3 ) and in urine (154). A selective estimation of Be with morin crayon on the ring oven is described in detail for 0.1-0.2 pg of Be. Interferences are also reported (729). A recent review of fluorornetric methods for boron has been compiled with 26 references (687). Benzoin is used as the reagent in a majority of the references cited and is also used in the determination of trace amounts of' B in soils (516). Several reagents for boron require concentrated sulfuric acid solutions. The acetylsalicylic acid coinplex with boron in concentrated sulfuric acid has an excitation max. a t 365 nni and an emission max. at 410 nm; the reagent emission is a t 430 nm and will detect down to 0.01 pg/ml of boron l(517). Another new reagent for B, 1, l'-immodi(6-chloroanthraquinone) is heated t o about 125 "C in 96% sulfuric acid to form the complex and has a sensitivity (Sandell) of 0.5 ng of B per square cm (231). Quinizarin also requires 91-96% sulfuric acid and will detect 0.01 ppm with the emission a t 595 nm and excitation a t 365 nm (282). Dibenzoylmethane in an ether-concentrated sulfuric acid mixture will detect 0.5 ng B/ml (420). A procedure for the determination of B in high purity tin has been developed with the use of benzoin as the reagent (391) ; the boron content is often near 5 X 10-595. I n this connection it should be noted that new platinum crucibles often contain B, unless ordered B free, and must
ANALYTICAL CHEMISTRY, VOL. 42, NO, 5, APRIL 1970
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be treated with sufficient alkaline fusions to render them usable for B analysis (132).
Calcium, Magnesium, and Lithium. Two new fluorometric reagents have been suggested for calcium. T h e 1:l complex of C a with 8-quinolylhydrazone of 8-hydroxyquinaldehyde at a p H of 11-13 is highly fluorescent and is sensitive to 0.02 pg of Ca/5 ml (80, 83-86). Tenfold concentrations of Rlg, Ba, Sr do not interfere with this reagent but many other ions decrease the fluorescence and must be removed. Another new reagent for calcium, bis (dicarboxymethylaminomethyl) - 2,6dihydroxynaphthalene, forms a 2 : 1 metal to ligand complex with Ba, Ca, Sr, and Mg with a maximum fluorescence a t pH 11.7 and 445 nm and an excitation max. at 385 nm. Aluminum and Be fluoresce with the reagent at a n optimum pH 5.8 and 5.2, respectively (100). This reagent is sensitive to 10 ng of Ca and less than 20 ng of Mg, 70 of Sr, and 150 of Ba, do not interfere with 300 ng of Ca. Calcein and calcein type indicators have been the subject of many papers. The calcein method has been automated for micro amounts of Ca in biological material (71) and also for both calcium and phosphorus (348). The complexes with calcein have been separated chromatographically (149). Methods have been described for the use of calcein for the determination of Ca in serum and urine (549), for the determination of microamounts of Ca in tissue (267), for ultramicro amounts of Ca in plasma (S93), for ultramicro amounts of Ca in biological fluids (440) and other biological materials (698). Various chelatometric indicators have been tested for the determination of Ca (157). New calcein type indicators using glycine in place of imidoacetic acid have been prepared and tested. Bis-4’,j’-N,N-glycinemethylene-2’,7’-diclilorofluorescein is a good reagent for Ca and also for the titration of Cu with E D T A (60). A number of substituted coumarins have been prepared and tested as nietallo fluorescent indicators (292). A structural study of the methyleneiminodiacetic acid (R) derivatives of 7-hydroxycoumariiis shows that the R group goes to the 8 position and if the 8 position is occupied it goes to the 6 position (559). Of the available fluorometric reagents for Mg ; 2,3 bis-(salicylideneamino) benzofuran is rated as the most sensitive; details are also given for the determination of up to 2 ppm of hlg in X i (143). The fluorescent quantum efficiencies of some useful chelates of ?\lg have been determined (142). Lumomagnesoii [ (2-hydroxy-3-sulfo-5chloropheny1azo)-barbituric acid] has been used for the determination of trace quantities of hfg in serum and 60R
urine (249); calcein (756) and 8-quinolinol are also used for M g in biological materials (494,601). Experiments have shown that calcium gluconate and calcium glucogalactogluconate have no significant effect on the determination of M g in urine with 2,2‘-dihydroxyazobenzene (479). This same reagent has been used for the determination of M g in soils (651). Disubstituted methanes are being studied as fluorometric reagents and bis(benzothiazo1)methane has been found satisfactory as a reagent for Li and Zn (556). With this reagent, zinc can be detected as low as 2 ppb with an emission at 450 nm and excitation at 410 nm. Na, K, and Li ions do not fluoresce v i t h this reagent. Lithium has been determined also using 8quinolinol as a reagent (413). Cadmium and Zinc. Reviews of the fluorometric methods have been given for zinc (693) and cadmium (688). The conditioiis for the determination of Zn and Cd with 8-(p-tosylsulfonamido) quinoline have been reviewed and the interferences given (668). This reagent has been used to determine traces of Zn in titanium dioxide (759). An analysis of zinc in biological fluids uses the 8-quinolinol method (405). A new reagent for Zn and Li is discussed above under Li. A new reagent, 3,3’-bis-[bis(carboxymethyl)aminomethyl] -4,4’-dihydroxy-trans-stilbene is suggested as a highly selective reagent for Cd; the emission maximum is a t 440 nm with the excitation at 360 nm. The effects of 27 cations and 15 anions have been determined (99).
the most sensitive and showed a n optimum p H of 8-8.5. The addition of sodium diethyldithiocarbamate increased the sensitivity to 1-2 ng Cu/5 ml (86). The clear blue fluorescence, 400500 nm, of 1-amino-(N,h7-dicarboxymethyl)-2-naphthol-4 sulfonic acid is quenched by the divalent ions of Cu, Ni, and Co and may be used as an end point indicator for the titration of Cu with E D T A (670). Another good indicator for the titration of Cu with E D T A is the glycine-calcein type mentioned in the section on calcium and described in reference 60. Several fluorometric methods for the determination of silver have been reported. One reagent, 2,3-naphthotriazole is reported as satisfactory for gravimetric, spectrophotometric, and fluorometric analysis. The fluorometric method gives a linear response from 0.025-0.1 fig Ag/ml (730). Small amounts of Ag (0.110 pg) can be determined by measurement of the fluorescence a t 485 nm obtained on the addition of S20,2ions to an aqueous solution, p H 1.53.5 containing silver ions and 8quinolinol-5 sulfonic acid. The fluorescence intensity is linear from 12.5 ppb to 5 ppm and most metal ions do not interfere; Cu, Hg, and Pd quench the fluorescence and Zr and Hf form strongly fluorescing chelates. The fluorescence is postulated to be due to an Ag(I11) complex (555). I n 8-9N sulfuric acid solution containing KBr, Rhodamine S (498) and Rhodamine 6Zh(6G) (664) and butylrhodamine (598) form a silver complex which is extracted into benzene and the fluorescence measured a t 550-580 nm. Silver Copper, Silver, Mercury, and Gold. also may be determined by titration of A new specific reagent, 1,1,3-tricyanothe nitrate in gelatin solution under uv 2-amino-1-propene produces a n inlight with K I at a p H 4-6 using calcein tense yellow fluorescence with Cu(I1) blue as an indicator to the disappearance a t a p H of 7.5-8.9 and is useful for of the blue fluorescence or to the ap0.015-0.6 pg Cu/ml. Directions for pearance of the fluorescence a t p H 6. the preparation of the reagent and the The recoveries are reported as quantitadeterniinatioii of copper in biological tive (185). Submicrogram amounts of materials are described (537). I n consilver to 4 ppb can be determined by the nection with this reagent a paper on quenching of the fluorescence of a mixcuproproteins by the same authors is ture of eosin and 1,lO-phenanthroliiie of interest (262). The red fluorescence (178). Trace amounts of silver may produced on the addition of thiamine to also be determined by the catalytic Cu(1) may be used as a spot reaction or effect on the chemiluminescence of the may be extracted with isoamylalcohol. lucigenin-H202 system; conditions and The test is sensitive to 6 pg of copper/ interferences have been given also 0.5 ml in solution or 0.3 pg as a spot on (53). The fluorescence analysis filter paper (749). Lumocupfera-benzamido-4-dimethylamino- methods for mercury are very similar to roii, those for silver. h mercury(I1)-Rhodcinnamic acid, in acetone-water solution amine B complex is extracted from a at pH 7-8 produces a bright green 1-25 HC1-KBr solution with a 1:1 misfluorescence with Cu(I1) (359). The ture of benzene and dioxane (198). The cupric ion also forms a fluorescent sensitivity is 1.6 ng Hg/ml; trivalent complex with N-(p-hydroxyethy1)ions of Ga, Au, T1, and Sn (IV) interfere. anabasine which has an emission at K i t h Rhodamine S the Hg complex is 420-510 nm and a linear intensity of extracted v i t h benzene from 9 N 0.1-1 wg/ml Cu at p H 9-12 (658). Of H2S04 which is 0.4N with KBr (302). three compounds used in a comparative Mercury can be directly titrated with study of the kinetic determination of E D T A a t pH 6 with “zincoii” as a traces of Cu, benzamido (p-dimethylmetallofluorescent indicator (630). benzylidene) acetic acid was rated as
ANALYTICAL CHEMISTRY, VOL. 42, NO. 5, APRIL 1970
Zinc, Cd, Pb, and M n are also titrated if present. Zincon is listed in Eastman Organic Chemicals 1966 Catalog as 0-{ 2- [ a-(2-hydroxy-5-sulfophenylazo) benzilidenelhydrazine ] benzoic acid, sodium salt. The Rhodamine B method for gold mentioned in the 1968 review has now been published (412). Other authors have reported on the butyl ester of Rhodamine B as a reagent for gold. The gold complex is extracted from a 12.6iV sulfuric acid solution with benzene and the benzene layer measured at about 565 nm (515). Gallium, Indium, and Thallium. A comparative study of Rhodamine S, salicylidene-o-aminophenol, morin, 8quinolinol, and lumogallion (2,2’4’trihydroxy - 5 - chloroazobenzene 3sulfonic acid, sodium salt) as reagents for t h e determination of G a led t o t h e conclusion that lumogallion was the most satisfactory for the fluorometric and Rhodamine S for the colorimetric measuremmt (596). Another group used these same reagents and rated morin a t the top (35). I t is possible that the lumigallion complex was extracted in one case and not in the other or that there was a temperature difference when morin was used. The extraction of the Rhodaniine 6G complexes of Ga and I n with benzene has been the object of a study where it was found that the extraction of the chloride complex of Ga was maximum in 631 H2S04 and 1J1 HC1 a t a G a ratio of 1: 2500; but in a ratio of 1: 10,000 6-11 HC1 !vas the best medium. The maximum extraction of I n was from 0.3-11 H B r and 531 H2S04 (6). The formulas for some colorimetric and fluorometric reagents of Ga and Be such as beryllion I1 and I V and lumogallion are included in a publication on a comparison of reagents (565). The compound 2,2‘pyridylbenzimidazole serves as a fluorometric reagent for Zn in the range of 15-800 ng/ml, for Ga 70-700 ng/ml and I n 110-1000 ngjnil (44). Interferences are avoided by extraction procedures. The sensitivity of lumogallion as a colorimetric reagent for Ga is 0.05-0.2 pg/ml and as a fluorescence reagent is 2 ng/ml in water and 1 ng/ml by amyl alcohol extraction. The fluorescence emission max for the Ga complex in water is 590 nm and in amyl alcohol is 600 nm. The max. absorption is 500 nm (417). The Ga complex with o,o‘-dihydroxy azo compounds a t low p H values, 3-4, is a 1: 1 ratio and is fluormcent; at p H values 6-7 the ratio is 1:2 and is not fluorescent (465). This same ratio is true also for quercetin a t comparable p H values (455). Another reagent suggested for Ga, ohydroxyphenylbenzoxazole, will deterKO interference was mine 0.6 pg. caused by 100-fold excess of Ag, Hg, hfg, Cr, Ba, A l i i , and 20-fold excess of
-
Zn, Cd, Zr, Co, and P b (284). The procedure for the use of the method with 8-quinolinol and extraction in chloroform has been outlined for the determination of traces of G a in silicate rocks and flue dust (153). Trace amounts of metal ions which form stable complexes with E D T A at pH 4 were titrated with morin as an indicator. Back titration of excess E D T A with Ga(II1) and an instramental end-point detection were employed for metal ions which are not normally determined by fluorometric methods (736). The continuous monitoring of the change in fluorescence intensity throughout this titration also allowed mixtures of Ga and I n to be determined when triethylenetetraminehexaacetic acid and EDTA were used as double titrants. A new reagent for I n is known as pyronine G, dimethy1-[6-(dimethylamino)xanthen-3-ylidene] ammonium chloride. The optimum reaction medium contains 2.5-3-12 HBr and 0.4-2.8 m M reagent. The I n complex is extracted into 1: 3 dry benzene-acetone. Two moles of reagent react with one of In. The concentration of I n in the extract may be determined either by absorptiometric or fluorometric measurement. The fluorescence emission is 582-588 nm. The lower limit of detection reported is 5 pg/ml (70). After a preliminary extraction, from 4-6N HBr with butylacetate, I n may be complexed with Rhodamine S and extracted from 4-6h’ H2S04 with 9:1, CsHs-HOAc and determined with a sensitivity of 0.5-1 pg/ml(446). Thallium may be determined quantitatively in a sodium iodide crystal with a simple procedure. The crystal (0.2 gram) is heated with 3 ml of concentrated nitric acid t o remove the iodine then with concentrated hydrochloric acid to remove the nitric acid and then with hydrazine to reduce Tl(II1) to Tl(1). The excess hydrazine chloride is removed and the fluorescence measured with the excitation at 245 n m with a sensitivity of 0.05 pg/ml (579). Thallium may also be determined in 7 N HCl to 0.03 pg of T1/0.2 ml a t room temperature and 0.004 pg/0.2 ml a t -196 “C. I n 7 N HBr the sensitivity is 0.005 pg T1/0.2 ml (64, 632). Antimony, Bismuth, and Lead. These metals and several others may be determined b y t h e fluorescence of their chlorides or bromides at liquid nitrogen temperatures. T h e elements, Sb, Bi, Ce, P b , T e , T1, Sn, Cu, U detection limits a n d excitation a n d emission wavelengths are recorded (344). A general discussion of new applications of luminescence analysis of inorganic substances gives a similar Table (87). The optimum conditions for the determination of Sh as a bromide (343) and P b and Bi as chlorides have been established (346) and excitation
.and mission curves are included. The analysis for Bi has been accomplished with Rhodamine B b y extracting the Bi complex a t p H 5 with a 3: 2 isobutyl ketone-benzene mixture (624), and also with flavonol-2’-sulfonic acid (748). Both of these references report absorption measurements but fluorescence measurements would just as easily apply. Both Rhodamine 6Zh and Rhodamine S were tested as fluorometric reagents for Sb. The 6Zh(6G) was found to be more sensitive, 0.1 pg/ml. The complex is extracted from a 6N H2S04and 0.5iV chloride medium (303). Luniinol is used for the chemiluminescent determination of Sb as the SbCls- ion a t a p H of 11 to 12. I n this case Sb(II1) is oxidized to Sb(V) with h’aX02 and the excess N a X 0 2 is destroyed with urea. The r e a h o n is sensitive to 0.05 pg Yb/ml with the luminescence measured on a photographic plate (353). Iron, Cobalt, Nickel, Chromium, and Manganese. T h e catalytic effect of iron on t h e chemiluminescence of luminol in t h e presence of H202 a n d triethylenetetramine has been further studied and conditions were outlined for t h e analysis of small amounts of iron (29). Iron a t ppb levels can be determined by its quenching effect on the luminescence of terpyridine (194). A general article on the determination of cations by quenching of luminescence shows that traces of Fe, Co, Xi, %In, and V can be determined in this manner (369). The oxidation of stilbexone with H202 is accelerated by Fe(II1) and 0.005 pg of Fe/5 nil can be determined by the decrease in fluorescence (376). A series of N-carboxylalkyl derivatives of aminonaphthalene sulfonic acid have been synthesized and two have been shown to be good reagents for Fe, Co, and C u through fluorescence quenching (670). Cobalt in ng amounts has been measured by the quenching of fluorescence of .-Il-I’UBR (147). Likewise Ni in ng amounts and Co in pg amounts decrease the fluorescence of the A1-PAN complex in a linear fashion (572). Two possible fluorescence reagents for Cr(II1) have been reported. These are t,he measurements of the fluorescence or phosphorescence of tris-(diethyldithiocarbamat0)-Cr(II1) (108) and the decrease in the fluorescence of triazinylstilbexone caused by Cr(II1) (671). Two luminescent methods for manganese analysis depend upon the fluorescence of a solid matrix after the addition of a drop of hIn solution. One method determines as low as 0.1 pg/ml/gram of matrix where the matrix is a Li-Mg-tungstate phosphor. The excitation is near uv and the emission is 668 n m (82). The other method with a matrix of Sb204 10 mole of B20a will determine 0.01 ng-1 pg of h l n ; the
+
ANALYTICAL CHEMISTRY, VOL. 42, NO, 5, APRIL 1970
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excitation used was the 313 n m Hg radiation (9). Another procedure depends upon the catalytic oxidation of 8-quinolinol to produce a highly fluorescent product where the response is linear for 2.5 ppb to 2.5 ppm of h l n (485).
Germanium, Thorium, Hafnium, and Zirconium. T h e procedure for
t h e fluorometric determination of Ge in coal ash with Rezarson has a sensitivity of 0.02 pg Ge/5 ml (401). The reagent Rezarson (2,2',4'-trihydroxy3-arseno-5-chloroazobenzene) and its use have been adequately discussed (400). An article on the analysis of variable valence ions b y the luminescence of the chlorides and bromides a t low temperatures includes Sn(I1) and (IV), In, Ga, Ge(IV), and Te(1V) (51). Both morin in 50-7070 methanol (SO) and quercetin (31) have been used for the determination of T h . The quantum yield for T h morin is given and quercetin is said to detect 2.5 pg/5 ml. Quercetin is reported to permit the analysis of Hf in the presence of Zr. I n 9-11 HC104 containing 2.5y0 ethanol Zr does not form a fluorescent complex with quercetin but the Hf complex is intensely fluorescent a t 505 nm when excited a t 340 nm (96). A linear result is obtained with 1 X 10-6J1quercetin and 1-20 pg of Hf which is unaffected by a five-fold excess of Zr. hlorin is used to determine 0.002O.O04Qj, Zr (514). An article on the luminescence and photochemical properties of complexes of T h with Schiff bases is of interest for prospective methods of analysis (480).
Vanadium, Tungsten, Niobium, Iridium, and Molybdenum. Several additional dyes have been tested for t h e catalytic fluorescent determination of vanadium but none were found superior to trypan red (69). The use of flavonol has been applied to the determination of tungsten in almost pure tungsten and in alloys (72). A new group of 2,7-bisazo derivatives of chrcmotropic acid was synthesized and the reactions with niobium were studied. The reaction occurs with il'b on the o,o' dihydroxy group in strongly acid solution in a 1: 1 ratio ( 7 ) . A new reagent, 2,2'2"-terpyridine has been suggested for iridium. The chelate has an excitation of 365 nm and emission a t 520 nm which decreases rapidly in intensity from 10' to 70 "C. The analytical curve is linear from 2 to 20 ppm; the degrees of error caused by Ru, Rh, P t , Xi, and Fe are also given (193). 110lybdenum may be determined by an absorption fluorometric method b y titrating Na2;lIo04with lead nitrate and with primulene as an indicator (18). Uranium. A review of t h e fluorescence analysis methods for uranium includes 41 references (692). The direct 62 R
measurement of the fluorescence intensity of uranyl nitrate in 20% tributyl phosphate -80% S p t h i n e (hydrogenated kerosene) gave a linear response for 0.01-lmg/ml in liquid NSwith front surface reading (160). A spot test for 1 pg of U is described where a neutral uranyl solution is added to 0.5 gram of 1: 1 Xa3P2Oio-CaSO42H20 mixture (347). Traces of U in thorium nitrate may be separated by ethyl acetate extraction and removal of T h from the extracted liquid by scrubbing with EDTA- aluminum nitrate solution (152, 640). h process for separating U from phosphate rock uses a 20 ml concentrated HC1-4 ml 30% H202mixture for extraction (663). A 95-page book on luminescence methods for U has been published in Xoscow (159). Lanthanides (Rare Earths). A ninepage review gives a summation of the papers presented a t the Budapest Conference on Luminescence (192). The individual reports pertinent to this review are given below. One of the most exciting developments in the analysis of the lanthanides has been the growth in popularity in the use of cathode rays and x-rays to generate a fluorescence emission between 200 and 780 nm. The application of the electron probe as an excitation source and a description of the arrangement of a photoelectric photometer to measure the emission has been given (349). A h a l p sis was made a t about 50 ppb with a limit of gram. Gadolinium, E u , and Sm have been determined in metallic U by spark excited radiation of luminophores based on Y203 for Gd and Yvo4 for E u and Sm (19). The determination of traces of lathanides in Y and La oxides (673), and of Eu in Y and Gd niobates and tantalates has been accomplished by cathode luminescence (89). I n a discussion on cathode luminescence as an analytical technique for determination of the rare earths in yttria the conclusion is reached that electron excitation is more sensitive than uv or x-ray excitation and in a good cathode luminescence host such as Y203 all rare earths are excited efficiently and with relative uniformity (735). Qualitative studies of rare earths in Y@3 show that 1-50 ppm of Eu, emission 553 nm and Ho, emission 550 nm are detected (381). When YOCl is used as a host for cathode ray analysis it undergoes irreversible change but does serve as a good system with rare earths for visible emission (605). A method has been proposed for the simultaneous determination of Gd, Tb, Dy, and E u in Y203,by the lines emitted a t 311, 543, 5i1, and 602 nm respectively, on excitation with a spark discharge between W electrodes (423). Cathode luminescence has many interesting geological applications in pe-
ANALYTICAL CHEMISTRY, VOL. 42, NO. 5, APRIL 1970
trology, mineralogy and paleontology which are outlined i n a brief historical survey (623). Rare earth analysis by x-ray excited optical fluorescence was the topic of a nine-page report a t a 1967 conference on applications of x-ray analysis. The emitted fluorescence from the rare earths ranges from 310 nm to 980 nm on irradiation with x-rays from a Cr target and 1-100 ppb of 11 lanthanides on Y203 or Gd2O3matrixes were determined (102). Optical fluorescence excited by x-ray has shown detection limits greater than mass spectrometry, emission spectrography, and neutron activation (128). The sensitivity is matrix dependent and detection limits of 11 lanthanides in Y203 and Gd2O3 range from 0.002 to 10 ppm (308). Details are given for detection of the La series as impurities in ionic materials and doped single crystals (591). The technique of x-ray excited optical fluorescence has also been applied to the determination of ppm of rare earth impurities (148). I n this case a tungsten target x-ray tube was used as the source. The fluorescence emission of Pr was measured a t 502.8 nm and E u a t 611.5 nm. Short wave uv sources are used for the analysis of a number of lanthanides. Cerium(1V) is reduced to Ce(II1) with Ti(II1) sulfate and excited a t 260 n m with a germicidal lamp to produce the 355 line of Ce (140). This reduction of Ce(1V) is also acconiplished with hydroxylamine hydrochloride in the determination of Ce in high purity rare earth oxides (208). I n yttrium oxide powder only Pr, Sm, E u , Gd, T b , Dy, Ho, Er, and T m were found to give sufficent emission 315-630 nm for analysis when excited a t 232-448 nm (482). Europium has been determined in borax glass a t a concentration of 0.0012% with a xenon arc light source and a laboratory-constructed fluorometer (534). Samarium in cerium dioxide has been determined by forming a crystal phosphor with PbS04 and LiF and measuring the radiation when excited with a high pressure mercury vapor lamp (81). Thulium has also been determined by the synthetic phosphor method (341). h method has been devised for the determination of sin of the rare earths as impurities in yttrium oxide in which the sample is treated Ivith NH4V03 and compared to standards containing these elements (520). Europium and S m have been determined by their fluorescence in complexes with P-diketones and organic bases (358, 424), or in other complexes (357, 607). Cerium (111) may be determined in the presence of T h with a sensitivity of 0.25 pg/5 ml by the fluorescence a t 596 nm of its complex with 4-[(2,4-dihydroxyphenyl)azo]-3-
hydroxy-1-naphthalenesulfonic acid (295). Yttrium, La, Lu, and Sc form a yellow green (560 nm) fluorescent complex with 5,7-dibrornohydroxyquinoline under longwave u v ; other rare earths do not fluoresce. The yttrium complex is extracted by benzene a t a lower p H than is La (370). Bis [ 1(2)-p~~idyl-3-methyl-5pyrazolonyl]-4,4'-methane is reported as a reagent for T b and Dy. The T b emission is a t 549.5 nm and is sensitive to 10-*-10-3~0 and the D y emission is at 574.8 n m and is sensitive to 10-I10-2yo (105). h study of the p-diketone ligands showed that the fluorinated ligands gave better sensitivity than the unfluorinated and the best found was 2-thenoyltrifluoroacetone 1% in D M F for concentrations in the order of 10-851 T b and Eu. For concentrations greater than 10-3J1 T b and E u may be determined b y directly irradiating the chlorides dissolved in D M F . The excitation and emission wavelength for T b are 370 and 545 nm and for E u are 390 and 615 nm (49). The determination of E u with the trioctylphosphine adduct of benzoyltrifluoroacetone is reported in the order of 5 x 10-7k! (608). Extensive studies have been reported for the reaction of Sc with the usual fluorometric reagents for trivalent ions. K t h the hydroxyanthraquinones the usual Sc to ligand ratio is 1: 1 and the optimum p H is 5-53 (463). With the 2,2'-dihydroxy type azo compounds, the ratio of Sc t o ligand is 1 : 1 in the more acid medium and 1 : 2 in the less acid ( 2 2 ) . K i t h the polyhydroxyflavones as morin and quercertin a 1:1 ratio is prevalent (464). The fluorescence intensity of Sc complexes with the 5,7-dihalo-8-quinolinols in CHCll are in the order: dichloro > dibromo > diodo-8-quinolinol. I t is also stated that 5,7-dichloro-8-quinolinol is more sensitive in the method of extraction described than is the 8-quinolinol method for Sc (467). rlnother extraction method for Sc determines 0.01 pg/ ml with a complex formed with morin and antipyrene a t p H 3.3-3.4 fluorometrically and 0.1 pglnil photometrically. The molar ratio of Sc-morinantipyrene-C1O4- in the complex in HC104 solution is 1:1 : 3 : 1 (462). iinother reagent, salicylsemicarbazid is reported to determine 0.03y0 Sc with a relative deviation of about 10% (597). Nonmetals. -4 20-page review on analytical methods for selenium contains 73 references and indicates t h a t for t h e analysis of submicrogram amounts of Se in biological materials fluorescence and radioactivation are the choice (724). A number of authors have reported on the application of 3,3-diaminobenzidine (DAB) as a reagent for Se. The complex may be measured spectrophotometrically at 220 n m or fluorometrically a t 550-600 nm
with a n excitation a t 420 n m and has a sensitivity of 0.01-1 ppm Se in biological material (130). The complex may be extracted with toluene and measured b y its fluorescence (554). Some workers prefer a dithiol extraction of Se into CCL before using the DAB reagent (364). Selenium has been determined with DAB in hard tissues and bones after i t is isolated b y distillation of SeBrr (409). Less than 0.1 pg Se/l. has been determined using the toluene extraction of the complex (402). One article lists 13 additional reagents for the spectrophotometric and fluorometric determination of Se (615). The trace of Se in sulfate reagents is also detected with D,4B (264). Selenium has also been determined in submicrogram amounts in a variety of materials with 2,3-diaminonaphthalene (DAW)as the fluorometric reagent-for example: 0.005 ppm in dried plants (255), 0.01 ppm in biological materials (I@), dietary mineral mixtures 0.25 pg/gram (378). From 3 to 5 ng of Se has been determined in milk with an error of 1 1 . 2 ng (358). The method used for the determination of Se in agricultural materials where the complex is extracted into cyclohexane has been described (277). A collaborative study of the DAK reagent has been reported (476). Dithizone has been used for the fluorometric determination of Se, and Rhodamine S for T e in a simultaneous determination of these elements (595). Selenium (65) and As (52) may be determined in very small amounts b y the fluorescence of their halides in HCl or HBr a t -77 "C and T e may be detected in 9 X HCl a t 0.02 ppm a t - 196 "C (542). The fluorometric methods for fluoride determination have been reviewed (689). The Th-morin method for fluoride analysis depends on the reduction of the fluorescence of this complex on the addition of fluoride. The result is a linear decrease up to 50% reduction in fluorescence intensity and agrees with results obtained with the fluoride electrode and with radioactive fluoride (666). The quenching of the fluorescence of the Zr-flavonol chelate (250) and the Al-PAK[ 1-(2-pyridylazo)2-naphthol] chelate (573) are both reported to give accurate results for fluoride analysis. The determination of sulfate by fluorometric or turbidometric methods has been reviewed with 22 references (690). The determination of cyanide with p-benzoquinone, N-chloro-p-henzoquinoimine, 2,5-dichloro-4-benzoquinone or o-(p-nitrobenzenesulfony1)quinone monooxime has been patented (242). The fluorescence and phosphorescence characteristics of 12 phenyl substituted silanes have been determined and their fluorometric assay is discussed (443). A method for de-
termination of the iodide ion is based on the quenching of the fluorescence of uranylacetate in sodium hydroxide solution (93). A fluorometric method for the determination of ammonia depends on its reaction with 2-oxoglutarate, water, and NADH2 in the presence of an enzyme, glutamate dehydrogenase to form KAD with a decrease in fluorescence (547). A chemiluminescent method for the determination of chloride ion in conductivity water is based on the fact that the C10- ion quantitatively increases the luminescence intensity of the luminol-H202 reaction (34). The test is sensitive to 0.5 pg of C1-/10 ml. Another method for chloride is the titration with Agf with dichlorofluorescein as an absorption indicator; the end point may be observed b y color or fluorescence (207). Hydrogen sulfide in the atmosphere may be determined to 0.2 ppb b y the decrease in fluorescence of fluorescein mercury acetate (26). This same reagent is used to determine sulfide ion 1-10 ng/ml of solution (233). A highly sensitive method for the determination of oxygen is based on its quenching effect on the phosphorescence of trypaflavine absorbed on silica gel; less than 4 x nm of oxygen can be detected (478). Trace amounts of ozone in the atmosphere may be determined by its oxidation of 9,lOdihydroacridine to acridine and measurement of the fluorescence a t 482 n m (722). An ethanol-acetic acid medium is used for this test which is about twice as sensitive as the phenolphthalein procedure. An automatic ozone analyzer which is based on the chemiluminescence of luminol is diagramed and described (158). ;1 chemiluminescence method for H202 sensitive to 10-8 mole/liter is based on the oxidation of luminol catalyzed by hemin in the presence of the Cu-triethanolamine complex (572). Chemiluminescence and pH Indicators. A 121-page survey of t h e recent literature on chemiluminescence covers t h e usual and also less familiar compounds, q u a n t u m efficiency, spectral distribution, and parameters affecting t h e reactions are also given (331). Another review from Poland is in two articles; the first covers 20 pages and 68 references (628) and the second, 29 pages and 117 references (627). A good general article shows how current theories have made possible the design of new chemiluminescent systems (531). A book in Russian on chemiluminescent techniques in chemical reactions has been translated into English (616). The third of a series of reports on the chemiluminescence of indok derivatives is from Japan but publihhed in English (648). The names of many new chemiluminescent compounds some of which
ANALYTICAL CHEMISTRY, VOL. 42, NO. 5, APRIL 1970
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are amazing because of the simplicity of their formulas and intensity of their luminescence, such as tet'rakis (dimethylamino) ethylene, have been published in pat'ents (122, 293, 586). The chemiluminescence of organic radical anions is also reported in a patent (532). A compound l,Pbenzo(g)phthalazinedione undergoes a reaction wit,h hydrogen peroxide similar to luminol but has a bright violet luminescence (733). Microcalorimetric investigat,ions have continued (163) and the quant,um yield of some luminol reactions have been determined (323). The chemiluminescence of oxidized oleic acid increases greatly in the presence of FeS04 (649). Various amines have been shown to increase the luminescence of luminol in the presence of H?Oz and h1n (326). Luminol-HeOz has been suggested as an indicator in the E D T A titration of the alkaline-earth elements (653). The enhancement of the chemiluminescence of the reaction between polyphenyls and ozone by Rhodamine 13 is thought to be caused by an energy transfer system (304). A method has been developed for the continuous measurement of chemiluminescent intensity obtained on the luminol-H20z mixture with changes in p H of the solution. The results showed that luminol consists of five components, four of vvhich are chemiluminescent (62). The relative quant>uni yields of siloxene with various oxidizing agents have been determined and permanganate, ceric sulfate, acid dichromate are rated as especially reactive ( 2 8 ) . The chemiluminescence of siloxeiie with Ce(1V) has been shown t,o be more intense in H2S04than in HC1 or HKOs (223). The chemistry of siloxene and other sheet-like silicon compounds has been reviewed with 32 references (270). The chemiluminescence of nitrogen monoxide on oxidation with oxygen or ozone has been suggested for monitoring of these gases and CO as air pollutant,s (1). A special fluorometer has been designed to measure the chemiluminescence produced from the reaction of bacteria with luciferin and luciferase. This system is said to be the first basic improvement in counting bacteria since the time of Pasteur (419). Two automated chemiluminescent systems have been devised for detecting microorganisms in water in concentrations 103-105/ml. These systems use alkaline lurninol with sodium perborate or sodium peroxypyrophosphate (474). Electrogenerated chemiluminescence is the subject of a general article on the emission from aromat'ic compounds in solution. Analytical applications dealing with both qualitative and quantitat'ive aspects are also suggested (41). Lumiiiol (183) and lucigenin (388) have both been studied with electrochemically generated luminescence. Car64R
bazole (562), isoindols (761),anthracene (189), and diphenylanthracene (139) have all been shown to produce electrogenerated chemiluminescence under various conditions of concentration and solvents. The behavior of fluorescent acidbase indicators has been studied in three mixed solvents, water-methanol, water-ethanol and water-acetone. Various concentrations of the organic solvents were used and the color and intensity changes at successive p H ranges are recorded (327). The use of 4-methylumbelliferone as an acid-base indicator with a midpoint transition a t p H 7.6 is reported. Both the anionic and neutral forms fluoresce in the blue with a 70% quantum yield (112). Four good fluorescent indicators for acidbase titrations have been found in a study of the methoxy and ethoxyquinolinols; the p H ranges and color changes are recorded (466). Luminol, pyrogallol, and lucigenin have successfully been used as chemiluminescent p H indicators in the analysis of turbid soil extracts (626) ORGANIC AND BIOLOGICAL
Research papers in this field continue to increase in number each year, and those that can be noted here are an ever smaller fraction of the total. Fortunately, the number of review publications is also increasing. Reviews have been published on fluorometry in food chemistry (174) and in air pollution research (565). Spectrofluorometry as a new method of functional analysis has been described in some detail (500), and the versatility of luminescence spectroscopy in technical analysis has been discussed (629). Both a book (754) and a lengthy review (740) have been published on the analytical applications of phosphorimetry. Bomen has reviewed fluorescence and luminescence in biology (77) and Hastings (266) has covered a similar area. Complicating factors in the luminescence of organic compounds have been discussed (225, 758), and tables of such luminescence have been included in the Landolt-Bornstein data (580). Fluorometry in clinical chemistry was treated in smaller reviews (254,659). Comprehensive chapters on the determination of phenylalanine and tyrosine in blood (542), the automated analysis of fluorescent substances ( 7 4 ) , and bioluminescence assay such as that for A T P (645) have appeared in recent volumes of a series on methods of biochemical analysis.
Hydrocarbons and Heterocycles. The luminescence characteristics of aromatic hydrocarbons at room temperature and at 77 O K continue to attract many workers, because of both their theoretical interest and the practi-
ANALYTICAL CHEMISTRY, VOL. 42, NO. 5, APRIL 1970
cal importance of the carcinogenicity of some of the polynuclear compounds. As an example of the latter, fluorometric procedures have been published for determining benzo [alpyrene in smoked foods (2877, beer (452), cigarette smoke (144), paraffin wax for food use (548), aqueous media containing 0.07M caffeine (175), and after thin-layer chromatography on silica gel G containing caffeine (617),in amounts of the order of 50 ng or less. Benzo[a]pyrene and many related cancer-producing polycyclics were simultaneously determined by combined chromatography-fluorometry, in petroleum products used for food packaging (633),in water, soil, and plant extracts (374),and in atmospheric dust (168, 352, 637, 638). Similar methods have been used to analyze for other related air pollutants, such as dibenzo [a, elpyrene (56),acridine (570), and phenylene-1-one and 7H-benz [ d , e]anthracene-7-one (181, 639). Vse of low-temperature fluorometry for the analysis of mixtures of polycyclic carcinogens has been reported (286); with monochromatic excitation and high-resolution fluorescence spectroscopy, it was possible to determine five such compounds simultaneously without chromatographic separations (385). Direct fluorometry of chromatographic spots, without elution, has been applied to anthracene and benzopyrenes (63) and to fluoranthrene and pyrene in wood preservatives (505). The fluorescent indicator adsorption method for determining the aromatics content of hydrocarbon mixtures was developed further (621) and was also applied to paraffins and chloroparaffins (721). Fluorescence spectra of anthracene and napththalene, as excited b y a giantpulse ruby laser, have been recorded (307). A comprehensive survey of fluorescence lifetimes was published (67). The fluorescence and phosphorescence of nine polynuclear aromatics in poly(methyl methacrylate) have been measured as a function of temperature (371). Fluorescence yield values were reported for 18 aromatic compounds in solution (145), and for tetraliii in cyclohexane solution as a function of wavelength (252). For anthracene, pyrene, fluoranthrene, and triphenylene in alcoholic solvents, the fluorescence yields were quenched most in primary alcohols and least in tertiary alcohols (216). Concentration quenching of various types has been discussed (ZOg), and a study of the quenching of pyrene and anthracene fluorescence by 14 electron-acceptors showed that charge transfer is an important mechanism (450). Luminescence spectra, in some cases a t 77 O K , have been measured for 10 phenanthrolines (496), 14 polycylic aza-aromatics in the solid state (499), several benzimidazoles in acid media (354), and fifteen pyrazolines (469).
The quasi-line phosphorescence spect r a of several aromatic hydrocarbons have been interpreted (396) and used for analytical purposes (190). Solventsensitized phosphorescence and related solvent effects have been studied for phenanthrene, napththalene-&, and carbazole (5); the temperature dependence of the phosphorescence of coronene in n-paraffins has been measured (226). An investigation of electroluminescence spectra for the determination of such compounds as anthracene and pyrene has been reported (202). Oxygenated Molecules. T h e fluorometric assay of ethanol in blood, using alcohol dehydrogenase a n d nicotinamide adenine dinucleotide (NAD) , has now been automated wit'h excellent precision and accuracy ( 1 7 9 ) . Another enzymic assay, applicable to ethanol and several other alcohols, is based on fluorometric determination of the hydrogen peroxide produced in the reaction with alcohol oxidase (246). Controlled osidation of a-glycols with periodic acid yields aldehydes, which can then be determined fluorometrically by condensation with 2,4-pentanedione, dimedone, or 2-amino-5-naphthol-7-sulfonic acid (567); similar methods can be used to analyze for olefins or other aldehyde precursors. Glycerol in plasma, determined by fluorometry of the XA4DH produced in the coupled glycerokinase-glycerophosphate dehydrogenase reaction, has been adapted to an automated method with good results (350). Sawicki and Carnes have coniparcd the sensitivity and select'ivity of four different spectrofluorometric procedures for determining 18 different aldehyde.: by condeiisat,ion with dimedone or related compounds (568),and have used anthrone as a fluorogeiiic reagent for the analysis of air pollutants such as acrylaldehyde and pyruvaldehyde (569). -kcrolein can also be determined hy reaction with m-aniinophenol and measurement of the fluorcscciit 7-hydrosyquinoliie produced ( k ) , and similar procedures can be used for many related substances including glucose a i d fructose. .hi enzymic method for low concentrations of glucose in urine, based 011 fluoronictry of t,he NAkllPHproduced, has been developed into a semiautomated ~ r o c e d u r e (575-577). For gluc o , ~in 1)lasnia: a different enzymic mct hod employs the reaction with glurose osidase which yields hydrogen permide, followed by perosidase hydrorylat ion of honiovanillic acid to yield :in intensely fluorescent product (506). Eiizyniic nicthods can also bc used to dett,rniine glucose and inulin simultancously in the ultraniiciro range (762). .\ldoses, ketoses, pentoses, and disaccharides react with aniline or 2-naphthylnmine in butanol solutioii containing phthalate, to give lroducts that
fluoresce at 510 and 520 nm, respectively, and are useful for quantitation (123). Invert sugar, glycerol, sorbitol, mannitol, and 2,a-butylene glycol were determined in less than microgram quantities in beverages, b y thin-layer chromat,ography followed by spraying with 4,5dichlorofluorescein and lead tetraacetate, and fluorometry of the spots (662). For the location and quantitation of 2-deosy sugars on chromatograms, 2-thiobarbituric acid and 4'-aminoacetophenone offer the greatest potential among the various fluorogenic reagents tested (571). The conversion of lactate to pyruvate by lactate dehydrogenase, and t'he accompanying change of reduced KAD fluorescence, can be used to determine either acid by the proper choice of reaction conditions (133); the same is true of 3-hydroxybutyrate and acetoacetate in the presence of 3-hydroxybutyrate dehydrogenase (217). hlalic acid was assayed b y spectrofluorometry aft,er condensation with @-naphthol (117) or with resorcinol (644), both in sulfuric acid solut~ioii; the latter method was used also for several substituted malic acids. The same resorcinol reagent' served for the fluorometric determination of sebacic acid, but, tartaric, malic, and barbituric acids interfere (228). assay for 4,5-dioxovaleric acid was based on t,he intense green fluorescence due to a benzoquinosaline derivative produced by coiidensation with 2,3Microdiaminonaphthalene (346). gram quantities of citrate were determined by a n enzymic cycling procedure and the fluorescence of the XhD product' ( d r y ) , and also by quenching of the fluoresceme of the flavonol-t,ungstate comples (251). A specific and sensitive procedure for oxalate is based on its quenching of the flavonol-zirconium chelate fluorescence ( 9 2 ) . Various workers have reported on the fluorometric determination of honiovanillic acid in body fluids and tissues, by means of st,epwise osidation with permanganate after separation of interferences (214, 425, 656). The fluorescence spectra of five derivatives of hydrosycinnamic acid have been recorded (744). Boric acid complexes were used in fluorometric analyses for dehydroacetic acid in foods (605) and for salicylic acid esters (606). Simultaneous determinatioiis of salicyclic, salicyluric, and gentisic acids in urine were achieved b y spectrofluorometry in p H 9 buffer (510). The change of fluorescence mit'h p H has been utilized to distinguish natural from synthetic t,annins (522). Under specified conditions in dilute amnioiiia solution, flavonols exhibit fluorescence (escitation and emission maxima are listed) but flavones do not (329). A new method for the determination of many acid anhydrides and acid chlorides is by reaction with 2-
carboxyisonitroso-acetanilide and measurement of the resulting fluorescence at 460 n m (169). Residual amounts of peryleiietetracarbosylic - 3,4:9,10dianhydride in pigmenh were determined by boiling in aqueous Sa,COS and fluorometry of the tetracarboxylate at 480 nm (420). Fluorescence methods for coumarin and related substances in plants (296, 377) have been described. Changes in fluorescence caused by various substituents in 7-hydroxycoumarins (umbelliferones) have been studied and correlated with t'he Hammet't sigma constants (604); 4-methylunibelliferone is a good fluorescent acid-base indicator, for which the titration curve midpoint comes a t p H 7.6 (112). The determination of serum triglycerides (by saponification, oxidation of the resulting glycerol to formaldehyde, condensation iyith acetylacetone in ammonium acetate solution, and measurement of the fluoresceiit lutidiiie derivative produced) has been automated (120, 443, 546) and various improvements have been proposed (68, 121, 134). F a t content in milk was determined fluorometrically after addition of Phosphine 3 R (104, 356). L-maturated lipids in tissues were localized by their fluorescence aft,er treatment with 9anthraldehyde (339). Vitamins. Seventeen vitamins present in multivitamin tablets were identified by circular thin-layer chromatography, after separat'ion into t h e water-soluble and fat-soluble groups by benzene extraction (265). In a detailed study of the fluorescence properties of vitamin -4 and the changes during photodecomposition, Kahan found that cyclohexane is the best solvent (320). Micromethods for vitamin in blood (166, 259) and liver (257) utilized deproteinization with ethanol, extraction with cyclohexane, and measurement of the fluorescence a t 480 nni under escitation at' about 340 nm. Vitaniiii 11 can be determiwd concurrently by escitation a t 295 n m and measuring the emission a t 340 rim (258). Other aspects of vit,aniin E fluorometry were studied by oxidative saponification of a-tocopheryl acetate with KOH-l\ln02, after which a strong fluorescence was observed at, 385 nm (655). Phosphorescence and fluorescence spectra of all-trans p-carotene and the 1,15'-cis isomer have been compared (116). Thiamine and its phosphate esters continue to be determined by the method of alkaline ferricyanide oxidation and fluorometry of the thiochrome produced (1f0,592, 7%'); this method has been applied, for example, to cheese (319). Thiamine and 2-(l-hydrosyethyl) thiamine can be assayed simultaneously b y similar procedures, but' using sodium hydroxide and then mer-
ANALYTICAL CHEMISTRY, VOL. 42, NO. 5, APRIL 1970
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curic chloride to destroy the thiamine differentially (437). For localization of thiamine in nerve tissue b y fluorescence microscopy, vapors of cyanogen bromide and ammonia were used for the conversion to thiochrome (661). Methods have been reported for the determination of riboflavine and various derivatives in blood (119), animal tissues (611), foods (191, 367), and feed mixtures (700) by means of intrinsic fluorescence (using permanganate oxidation to destroy interferences) or after photochemical conversion to lumiflavine. The adsorption and fluorescence spectra of lumiflavine have been studied in various solvents (365), and methods have been described for the separation of various photolysis products of riboflavine (366,681). Procedures continue to be published for pyridoxine and related compounds by oxidation with alkaline permanganate to pyridoxic acid, followed by lactonization in hot acid medium and measurenent of the fluorescence so produced (368, 583). A variety of vitamin Be compounds can be determined by means of this fluorescent lactone and the fluorescent cyanohydrin derivative (124, 389, 471). The effects of p H and temperature on the luminescence spectra of pyridoxal (719) and several related compounds (438, 439) have been reported. A simple and accurate analysis for ascorbic acid is based on its reduction of the quinone of dihydrosynaphthalene sulfonic acid a t p H 4 back to the diol form, which fluoresces strongly at 465 nm (290). Fluorometric determinations of folic acid in foods (230) and in urine (699) make use of permanganate oxidation to a fluorescent product. Amines. Reviews have been published on t h e fluorometric determination of catechol amines and their metabolites in urine (725) and of some biogenic amines and other neurochemical transmitters (539). The most favored method for adrenaline and noradrenaline consists of ferricyanide osidation to the fluorescent trihydrosyindole derivatives, with addition of a mild reductant to stabilize the intensity (160, 198, 386); the same principle has been used in several automated procedures (408, 418, 560). Among the improvements suggested were the introduction of sodium diethyldithiocarbamate (712) or P-mercaptoethanol (726) as the stabilizing agent. Differential determination of adrenaline and noradrenaline is possible if the fluorescence is stabilized with thioglycolate and with ascorbate, respectively, in two different aliquots (418, 713), or by using variations in p H (426) or excitation and emission peaks (595). A different fluorescence method is valuable in histochemistry, in which exposure of the frozen tissue section to formaldehyde 66R
causes the catechol amines to undergo closure of the side chain by a methylene bridge, and subsequent dehydrogenation to form fluorescent isoquinolines (184,538,558). A modification of the hydroxyindole technique, using iodine in place of ferricyanide as the oxidant, has been used for fluorometric determinations of several acid and alcohol metabolites of catechol amines (666). Metanephrine and normetanephrine (which are the 3methoxy derivatives) were assayed by the trihydroxyindole method, after separation from each other on cellulose phosphate columns (321). Fluorometry was also used for differential analysis of noradrenaline and a-methylnoradrenaline (718), and for determining noradrenaline, Bhydroxytryptamine, and dopamine in a single sample of brain tissue (20, 406). I n the fluorometric determination of histamineby condensat'ion witho-phthaldialdehyde, improvements (714) have been suggested which include preparing the reagent under anhydrous conditions (753), using phosphonic acid ion-eschange resin instead of cellulose phosphate for the preliminary purification (431), and minimizing interferences by control of p H and addition of citric acid (21). The phthalaldehyde method has been employed to detect traces of histamine in histidine (475), and to localize histamine in tissue sections (173, 314, 603);it has also been adapted to automation (550). Spermidine and histidine give fluorophores like that of histamine in this reaction, but their fluorescences are about 30-fold weaker on an equimolar basis (430,452). Serotonin (5-hydroxytryptamine) in 3N HC1 fluoresces strongly a t 550 nm under excitation a t 300 nm, and thus can be determined in tissues fluorometrically aft'er extraction with butanol from borate buffer (165, 360, 407, 473). Improvements in the method have been suggested (209, 714), and the literature on it has been reviewed (691). Other fluorometric procedures for serotonin involve condensation with o-phthaldialdehyde (676) or ninhydrin (373, 472, 527). Fluorophores are also generated by the reaction of formaldehyde with 5-hydroxydopamine ( 1 72) or with serotonin or other tryptamine, derivatives (312). Other publications describe the fluorescence properties of several indoles and aniline derivatives as a function of p H (91), t.he relative fluorescence intensities of various substituted indoles (39), and the fluorometric determination of 5-hydrosy-indoleacetic acid in urine by condensation with o-phthaldialdehyde (361) or by its intrinsic fluorescence a t 340 nm (340). Spermine was assayed fluorometrically by reaction with phenylacetaldehyde, after separation from spermidine (297), and a modified o-phthal-
ANALYTICAL CHEMISTRY, VOL. 42, NO. 5 , APRIL 1970
dialdehyde method for a-methyl-mtyramine and metaraminol was reported (526). A large number of primary aromatic amines have been determined fluorometrically by condensation with o-phthaldialdehyde (11) or with p-(dimethy1amino)benzaldehyde (451). The Rose Bengal complexes of certain aralkylamines were utilized for fluorescence analyses (380). Pesez and Bartos have continued their work on functional organic fluorometry with two papers, one of which is on the determination of primary and secondary alkylamines using 1,2naphthoquinone-4-sulfonic acid (501). Their other paper (502) describes fluorescence analyses for tertiary alkylamines with a, y-anhydroaconitic acid in acetic anhydride, for primary and secondary amines wit'h l-diniethylaminonaphthalene-5-sulfonyl chloride, for amino acids by condensation with acetylacetone and formaldehyde which produces dihydrolutidine derivatives, and for pentoses and steroids. Amino Acids and Proteins. Glycine and other amino acids and amines in submicrogram amounts were determined by reaction with 2,4-pentanedione and formaldehyde, in acetate buffer; the relative fluorescence intensities a t specified excitation and emission wavelengths were determined fGr 20 such compounds (566). Another highsensitivity method for amino acids was based on their reaction wit'h amino acid osidase and subsequent conversion of homovanillic acid into a highly fluorescent biphenyl derivative (239), Two-dimensional separat'ion of t'he fluorescent 5-dimethylaminonaphthalene-lsulfonyl derivatives of amino acids was achieved by combining chromatography with elect'rophoresis on a cellulose t,hin layer (23). Quantum yields for the intrinsic fluorescence of tyrosine, trypt'ophan, and phenylalaiiine were found by Chen to be smaller than the literature values (113). For tyrosine, a more intense fluorescence is produced by coupling with 1-nitroso-2-naphthol in the presence of nitrous acid; this method has been automated (275), adapted to dried blood on paper disks for clinical diagnosis (587), and improved with regard to interferences (15). Tryptophan in biological fluids has been determined directly by spectrofluorometry (720) or after conversion to highly fluorescent norharman (358, 390). Phosphorimetry was used by Hood and lf7inefordner for the determination of kynurenic acid in urine (285); 5-hydroxykynureiiiiie (337) and other tryptophan metabolites (55) were assayed by fluorometry. The phosphorescence and fluorescence of a series of phenylalanine and tryptophane derivatives have been studied (727). Because of the im-
portance of early detection of phenylketonuria, reports continue to appear on the use of the McCaman-Robins assay for phenylalanine in blood (61, 588, 672); this method, which uses the fluorescence produced in the reaction with ninhydrin and 1-leucyl-1-alanine, has been shortened (12) and adapted to automation on an ultramicro scale (14). Glutathione in biological materials has been analyzed b y fluorometry after condensation with o-phthaldialdehyde (421). Another fluorometric determination for glutathione or other sulfhydryl or disulfide compounds is based on the quenching of the fluorescence of mercurated fluorescein derivatives (705, 706, 747). Histidine was determined b y a modification of the histaminephthalaldehyde method, and the errors caused by many possible interferences were evaluated (13). Protein luminescence properties have been the subject of many papers, too numerous for consideration here. Attention is called instead to reviews on the fluorescence and phosphorescence of proteins and nucleic acids (355, 397), on fluorescence st'udies of protein struct'ure (114, 132, 64?), and on protein conformation (88, 170). Analyses reported for some proteins and other nitrogen compounds utilized the quenching of the fluorescence of tetramercurated fluorescein (746). Urea has been determined b y conversion to ammonia followed by enzymic reaction with a-ketoglutarate and measurement of the NAD produced (543). Another assay for urea (486)or related compounds (618) depends on the intense fluorescence obtained by reaction with the diosime of cyclohexa1ie-1,2-dione or other oximes. Uric acid was determined by treatment with uricase, producing hydrogen peroxide which was then measured by a reaction yieldiilg highly fluorescent indigo white (241). Enzymes, Nucleotides, a n d Nucleic Acids. R o t h has published a n extensive review of t h e fluorometric assay of enzymes, with 423 references, which includes several new methods (545), and Guilbault has written a shorter review (254). Biological staining with fluorescein or rhodamirie labeled enzymes has been proposed for the in situ localization of enzymic substrates (59), such as chitin by staining with fluorescent chitiiiase (58),as an extension of the well-known fluorescent antibody technique. A widely-used technique for the assay of hydrolytic enzymes is to add a nonfluorescent but fluorogenic substrate and measure the resulting luminescence. Such determinations have been reported for phosphatases with substrates of umbelliferone phosphate (248), of naphthyl AS-MX phosphate ($IO),of flavone 3-pyrophosphate (379), and of 3-0methyl-fluorescein phosphate (273,723);
for lipases with substrates of 4-methylumbelliferone heptanoate (247) or N methyl indoxyl myristate (238, 240) or fluorescein dilauric ester (429); for sulfatases with @-naphthol sulfate or 4-methylumbelliferone sulfate as substrate (238); for trypsinlike amidase with a substrate of the P-naphthylamine amide of a-benzoyl-Larginine (24); for leucine aminopeptidase with L leucyl-P-naphthylamine as substrate (544); for a-mannosidase with 4methylumbelliferyl - a - D - mannopyranoside as substrate (470); for @-galactosidase with fluorescein di-(Pg Jactopyranoside) as substrate (212); for cellulase with resorufin acetate as substrate ( 2 3 7 ) ; and for elastase with substrates of fluorescein-elastin (215, 536) or Reniazol brilliant blue-elastin (536). For some oxidative enzymes also, use has been made of an added fluorogenic compound. A prominent example is the determination of monoamine or diamine oxidases b y causing the hydrogen peroxide, produced in the enzymic reaction, to undergo reaction with added homovanillic acid (236, 630, 678) to form an inteiisely fluorescent product; in place of homovanillic acid, other workers have recommended p-hydroxyphenylacetic acid (235, 244) or kynuramine which gives 4-hydrosyquinoline in the enzymic reaction by osidative deamination (111, 167). Similar procedures were used for the assay of amino acid oxidases by fluorometric measurement of the amount of scopoletin destroyed by the peroxide produced (394) or by condensation of the pyruvic acid (formed enzymically from alanine) with 0-phenylenediamine to form fluorescent 2-hydroxy-3-methylquiiiosaline (?I 0), and also for xanthine oxidase using 2amino-4-hydroxypteridine as fluorogen (253), and for tyrosine hydrosylase by formation of dopa, 3,4-dihydroxyphenylalanine (44?). A general method for determining dehydrogenases or other enzymes requiring the participation of some form of nicotinamide adenine dinucleotide is by measuring the changes produced in the fluorescence of the KAI) compound. Such procedures were reported for glutathione reductase (582), triosephosphate isomerase (524),galactose-1-phosphate uridylyltransferase (65, 126), and for glucose-6-phosphate dehydrogenase (64, 553, 63.4) including automated methods for it and for 6-phosphogluconate dehydrogenase in red blood cells (660). Related fluorometric methods were described for metabolites in muscle (763). Other fluorometric procedures have been reported for creatine phosphokinase by the ninhydrin reaction (551, 739), for acetyl coenzyme A (8, 107), for catechol 0-methyl transferase (25), and for increased sensitivity in nicotinamide
nucleotide coenzymes analysis by t,he addition of ethanol to the reaction mixture (227). Fluorescence analyses have been described for the various forms of nicotinamide adenine dinucleotides (NAD, NADH, NADP, and KATIPH) in liver tissue ( H I ) , in Ehrlich ascites cells (6?6),and in plant material (555) and animal tissues (109) using reduction of resazurin to highly fluorescent resorufin. A specific assay for SXDPH was report,ed (57). Phosphorescence and fluorescence spectra of flavine coenzymes were studied (75), and phosphorescence was used to detect purine oligonucleotides in the nanogram range (529). I n the field of nucleic acids, a review was published on their luminescent cytochemistry (755). The method for determining DNA%and RNA by the fluorescence of the ethidium bromide complex has been automated with good results (708); the DNX content of seawater was measured by the fluorescence of the complex formed with diaminobenzoic acid dihydrochloride (285). The Acridine Orange complexes colitinue to be useful for the fluoroniet,ric deterniination of native IIKA arid denatured D S A in mixtures of the two (46,185). The assay of transfer RNA specific for phenylalanine in ye liver, or wheat germ, was accomplished by its intrinsic fluoresceiicc a t 440 nm (752), and phosphorescence characteristics were recorded for the transfer RNA specific for valine in Is. coli (269). Immunofluorescence. Appearance of the third edition of Nairn's book of fluorescent protein tracing (445) and t'wo other books in the U.S. on this field (10, 220) attests to its continued growth and importance. There have also been several foreign reviews on this powerful technique (210, 404, 455,717) which permits the detection and estimation of many molecular species or microorganisms used as antigens b y their immunochemical reaction with fluorescence-labeled specific antibodies followed by examination by fluorescence microscopy. Uses of this method iiiclude the localization of brain hexokinase (I%), of catalase in mammalian tissues (456),of ribonuclease and deoxyribonuclease in various tissues (455,?.$a),of prolactin (454), @-corticotropin (271),and growth hormone (453) in the pituitary, of insulin and insulin antibodies in diabetic eyes (728), of chorionic gonadotropin (325), of fibrin and plasma protein deposits in l)lacenta (434), and of intracellular immunoglobin (272). Some other applications reported were the diagnosis of Kilson's disease by the absence of ceruloplasniin in human liver cells (592), the detection of early syphilis (315), the assay of Salmonella in nonfat dry milk (553), the
ANALYTICAL CHEMISTRY, VOL. 42, NO. 5, APRIL 1970
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detection of Jiycoplasma mycoides, the infectious agent in bovine pleuropneumonia (414), the differentiation of various pat’hogenic species of Clostridia (47), the detection of botulinum E toxin (300), the determination of AB0 blood groups in human blood stains (449),and changes in Xenopus laevis toad hemoglobins from tadpole to adult (317 ) . -4 quantitative immunofluorescence assay for tissue sections was described (589)) the lack of specificity in commercial fluorescence-labeled ant isera was discussed (441), and the spectral data of commonly used fluorescent tracers mere reviewed (260). Steroids and Hormones. T h e fluorescence developed in st,eroids in sulfuric acid solution, which provides t h e basis for t h e fluorometric determinations, has been studied in terms of t h e changes due t o variations in chemical structure among cholesterol, estrogens, progesterones, androgens, and corticosteroids (612). The phosphorescence and fluorescence of 19 steroids on thin layer chromatograms sprayed wit,h 1 : l H z S O ~have been reported (137). A fluorometric method for sterols in egg noodles was validated by a collaborative study (540). The fluorescence assay of total bile acids can be modified to avoid (38) the need for prior hydrolysis of the conjugates (205). The fluorescence characteristics of prostaglandin El in 70% Hi304 were studied (213), and an analytical method was proposed for ACTH based on the production of corticosterone in hypophysectomized rats (613). Many papers continue to appear on the fluorometric analysis of corticosteroids by means of the usually 7 : 3 HZSO4-ethanol reagent (201, 906, 528) ; some deal with very lorn concentrations (161), the need for high-purity dichloromethane used in the preliminary estraction process (334, 716), and the role of other steroids as possible interferences (458). The procedure has been automated (305, 680). Simultaneous fluorometric determinations of cortisol and corticosterone have been accomplished by means of such methods as differential solvent estraction (4081, the great difference in their fluorescence intensity ratio in 6 : 4 HzS04-ethanol from that in 9 : 1 H2SOa-ethanol (274), metaperiodate oxidation to the corresponding 17 p-carbosylic acids, after which only the cortisol derivative is fluorescent (118 , and separation by thin layer chromatography (288). Two-dimensional thin layer chromatography gave a high dcgree of purification prior to the fluorescence assay of corticosterone in bird plasma ( 7 3 ) . -4 different kind of fluorometric met,hod for steroids was described, in which the steroid amidinohydrazones are condensed with phenanthraquinone (578). 68R
Testosterone and other androgens were deterniined fluorometrically after heating in 3 : 1 H2SOa-ethanol (289, 291, 636). Ittrich proposed dilution with alcoholic formaldehyde solution after the heating, for high sensitivity (301). I n anot,her method, the testosterone was transferred to the surface of a pellet’ of lithium hydroxide and the fluorescence was developed by heating (224,225). Spectrofluorometric methods for the determination of estrogens have been reviewed (643). Extensive use continues to be made of the Kober-Itt’rich method, in which the fluorescence is developed by heating in H2S04containing hydroquinone (95, 625). Different preliminary purification steps have been compared (635), and the method has been applied to pregnancy urine fractions (171). The effects of modified heating co~idit~ions and acid concentration on the assay of estrone, estradiol, and estriol were invehgated (97), and the escitation and emission spectra of these and other estrogens were recorded (581) Simplified fluorometric methods were studied and improvements suggested (127, 156). Automated procedures were described for the assay of urinary estrogens in nonpregnancy (646),pregnancy (444), and late pregnancy (709), and for ethinylestradiol in (336). pharmaceutical preparations The behavior of progest,erone in some fluorescence reactions was noted (7’45). The auxin plant-growth regulators and other indoles have been studied by spectrophosphorimetry (294, 642). Gibberellic acid was determined by fluorometry after treatment with stannous chloride in 85y0 H2SOa(221). Pharmaceuticals. T h e analysis of drugs in biological materials by spectrofluorometry has been reviewed ( 155). Phosphorimetric wavelengths and analytical procedures have been reported for 15 sulfonamides in blood (280). Aispirinand salicylic acid were determined by fluorometry after a gelfiltration separation (387). A new method for tetracycline employs conand version to anh!.drotetracycliiie formatioii of a highly fluorescent complex with et hanolic aluminum chloride (333); this was used in the analysis of mistures of tetracycline, chlortetracycline, and demethylchlortetracycline (332). -1 sensitive method for actinomycin D in serum was based on the fluorescence obtained by oxidation with alkaline hydrogen peroxide (1Q7), and solrent shifts in the intrinsic luminescence spectra of actinomycin D were interpreted (519). A fluorometric assay for isoniazid in serum utilizes reduction of the hydrazone to produce an intense fluorophore (585). Picornavirus inhibitors of the 2(a-hydroxybenzy1)benzimidazole type were determined by either the intrinsic I
ANALYTICAL CHEMISTRY, VOL. 42, NO. 5, APRIL 1970
fluorescence a t 322 nm or a more intense emission a t 375 nm after treatment with ammoniacal copper sulfate (481). Methaqualone in plasma was assayed by its fluorescence after reduction with lithium borohydride (98), and metapyrone was analyzed by reaction with cyanogen bromide and p-aminoacetophenone and fluorometry of the product (422). The intrinsic fluorescence of dapsone (4,4’-diaminodiphenyl sulfone) in anhydrous ethyl acetate was used for its determination in blood and urine (219); and benhepazone in clinical materials was also assayed by its own fluorescence at 410 nm in chloroform, under 395 nni excitation (446). The phosphorescence characteristics of 37 antimetabolites were measured, about half of which show analytical possibilities (561). Assay of 6-mercaptopurine in serum was reported, based on permanganate osidation to purine-6sulfonate and fluorometry a t 400 nm (195). The antitumor drug camptothecin was determined by its intense fluorescence at 434 nm (262), and daunomycin in biological samples was analyzed similarly by direct fluorometry a t 589 nm in solvent extracts (198, 489). Reduction with sodiuni hydrosulfite to a fluorescent product \vas used for the determination in serum of lapachol, which is 2-hydrosyl-3-(3-methyl-2-butenyl)-1,4-naphthoquinone, an antitumor and antimalarial drug which prevents osidative synthesiq of ATP (196). Fluorometric methods were reported for quinine (94) and morphine (94, 416, 657). Intrinsic fluorescence was used for the determination of harmane in botanicals (428), lysergic acid diethylamide and other indole alkaloids (17 ) , hydrastine and hydrastinine (743), ajmalicine (619), and physostigmine in tissue samples (667). Phenothiazine drugs in blood were assayed by spectrofluorometry after conversion to the sulfoxides (679). Thioridazine and mesoridazine were subjected to a brief permanganate oxidation before fluorometry a t 440 nm (484). Thin layer chromatography was utilized in the fluorescence analysis of medazepam in blood (384), and the specificity of a spectrofluorometric method for thiopental was demonstrated (146). Condensation with ophthalaldehyde served as the basis for the fluorescence assay of 4-(2-hydroxy3-isopropylaminoproposy)indole, a pblocking agent (483) in blood and urine. The muscle relaxant dantrolene, which is a hydantoin derivative, was analyzed by fluorometry a t 530 nm (281), and pyrimethamine by fluorometry a t 375 nm (311). Improvements were made in the separation processes preceding fluorescence analysis of quinidine in serum (263). Chromatographic procedures were described for the fluorometric determination of myocardial digoxin
and other digitalis compounds at autopsy (309). Agricultural Chemicals and ProdT h e analysis of aflatoxins, ucts. often by fluorometry of thin-layer chromatograms, continues as a very active field (278, 504, 683) because of the importance of avoiding ingestion of these substances which are produced by mold growth. Improvements have been proposed in the objective measurement of fluorescence intensity of such chromatograms (48, 521 , 610). The solid state fluorescence of various aflatoxins on silica gel (541) and the phosphorescence and fluorescence of aflatoxins E$ and GI (707) have been reported. Fluorescence methods have been used for rapid screening for aflatoxins in cottonseed (734), for determining the aflatoxin coiitent of peanuts sold for human consumption in Uganda (399), and in a screening procedure for aflatosin, ochratoxin, and zearalenoiie (182). Pesticide analysis by luminescence methods has been discussed (523), with emphasis on enzyme inhibition techniques (245). Kinetic fluoromet’ry was proposed for the analysis of organophosphorus cholinesterase inhibitors (243, 416). Benomyl, a carbamate pesticide, was determined by fluorometry after conversion to 2-aminobenzimidazole (495), and fluorescence quenching was used for the assay of triazine herbicides (206). The luminescence properties of the food antioxidants propyl gallate, butylated hydroxyanisole, and butylated hydroxytoluene were reported, and an analytical method presented for propyl gallate in lard by use of chloroform as solvent, which quenches the fluorescence of the other two antioxidants (382). Dulcin, p-ethosyphenylurea used as a sweetening agent for foods, was determined by fluorometry after hydrolysis and treatment with nitrous acid (696). Biphenyl and related compounds in citrus peel were assayed with the help of fluorometry (509), as also were scopolin and scopoletin in tobacco (741). The coumarin and psoralen derivatives responsible for the luminescence of expressed lime oil have been identified and their emission characteristics noted (385’). Glucobrassicin (an indole derivative) was determined by fluorometry after condensation with p-dimethylaminobenzaldehyde (518), and the resistance of corn to European corn borer was evaluated with aid of a spectrofluorometric assay of its 6-niethosy-2benzosazolinone content (7‘8). Miscellaneous. A review on automated fluorometric procedures (685) gives references for 34 biological compounds. The luniinesceiice of biopolymers and cells has also been reviewed (43). -2 description has been
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