Fluorometric Analysis

(69) Meissner, K. W., Van Veld,R. D., Ibid., 45, 903 (1955). (70) Mém. soc. roy. sci. Liége 14, 52-506 (1954). (71) Michel, P., “La Spectroscopie ...
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V O L U M E 28, NO. 4, A P R I L 1 9 5 6 Ibid., 26, 54-8 (1954). ,Meggers, W. F., J. Opt. SOC.Bmer. 44, 348-9 (1954). Meissner, K. W., Van Veld, R. D., Ibid., 45,903 (1955). Xdm. SOC. roy. sci. Liege 14, 52-506 (1954). Michel, P., “La Spectroscopie d’Emission,” Armand Colin,

Paris, 1953. Moore, C. E., Natl. Bur. Standards, Circ. 467 (in press). 2. Physilc. 137,575-82 (1954). Murakawa, K., Suwa, S., Noldeke, G., Steudel. A,, Ibid., 137,632-7 (1954) Olson, K. B., Heggen, G., Edwards, C. F., Gorham, L. W., Science 119, 772-3 (1954). Pery, A., Proc. Phys. SOC.( L o n d o n ) 67A, 181-5 (1954). Rasmussen, E., Middelboe, V., Z. Physik 141, 160-5 (1955). Rico, F. R., Anales real soc. espafi. fis. y putm ( M a d r i d ) 50,

185-200 (1954). Risberg, P., Arkib F y s i k 9,483-94 (1955). Rose, H. J., Jr., llurata, K. J., Carron, AI. K., Spectrochim. Acta 6, 161-8 (1954). Rosen, B., Rev. uninerselle m i n e s 9,445-54 (1953). Schuhknecht, W., Optilc 10,245-302 (1953). Schwartz, S., Astrophys. J . 119, 296-7 (1954). Scribner, B. F., SIeggers, W: F.. “Index to the Literature on Spectrochemical Analysis.” Part I. 1930-39, Part 11,

62 1 1940-45, Part 111, 1946-50, Am. Soc. Testing Materials, Philadelphia, Pa. Shenstone, 4.G., J . Opt. SOC.Amer. 44, 749-59 (1954). Spitz, E. W., Simmler, J. R., Field, B. D., Roberts. K. H.. Tuthill, S.M.,ANAL.CHEM.26, 304-7 (1954). Stanley, R. W., Dieke, G. H., J . Opt. SOC.A m e r . 45, 280-6 (1955). Stanley, R. W..Meggers, W. F., unpublished data. Steinberg, R. H., A p p l . Spectroscopy 7, 163-4 (1953). Steudel, A., Thulke. H., 2. Physilc 139, 239-42 (1954). Striganov, A. R., Korostyleva, L. d.. Dontsov, Y. P.. Z h u i Eksper. i. Teort. F i t . 28, 480-4 (1955~. Trans. Intern. Astron. L‘nion 9 (in press). Treanor, C. E., Phys. Rev. 95,1472-3 (1954). Vander Sluis, K. L., lIcXally, J. R., Jr.. J . O p t . SOC.Arner. 44, 87 (1984). I b i d . , 45, 65 (1955). Voinar. A . O., B i o k h i m i y a 18, 29-33 (1953). Wardakee, A. K., J . O p t . SOC.A m e r . 45, 354-5 (1955). Wilkinson, P. G., Ibid., 45, 862-7 (1955). Young, L. G., Spreadborough. B. E. J., Reid, P. lI.,Spectrochini S c t a 6 , 144-52 (1954).

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REVIEW OF FUNDAMENTAL DEVELOPMENTS IN

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Fluorometric Analysis CHARLES E. WHITE University o f Maryland, College Park, Md.

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his review covers the 2-year period since the previous review (91), from approximately August 1953 t o August 1955. In accordance with the policy of this journal. only a selected group of references are included. APPARATUS

Anyone concerned with selecting a source of ultraviolet light will find a n article by Doede and Walker ( 2 2 ) very helpful. These authors compare the ultraviolet radiation of about 50 commercial lamps. A rating is given for the lamps in five different parts of the spectrum from 2200 to 3800 A. A number of research groups during this period have built fluorometers with some special features. Galvanek and Morrison (39) report success with the use of the 360 BL type of fluoresrent lamp in a fluorometer for the determination of uranium. ;\ii electron multiplier phototube and a vacuum-tube voltmeter :we used in the detection unit. Kinser (68)has described another model of the Geological Survey transmission fluorometer in ahich he uses a General Electric R. P. 12 lamp for excitation and an endwindow photomultiplier tube in the detection unit. Another instrument, designed by Kelley and Hemphill ( 5 7 ) for the uranium determination, uses two AH4 lamps a t a 45” angle t o the sample and a 6199 photomultiplier tube. Florida and Davey (35’) have also given details for a photomultiplier fluorometer of the reflector type for the measurement of the fluorescence of Bolids in which the exciting source is a t about a 45” angle and the photomultiplier tube is a t a 90” angle with the sample. Lynch and Baumgardner ( 6 3 ) have designed a fluorescence photometer for use with both solids and liquids, in ahich both the known and unknown samples are irradiated: the light from each falls on separate phototubes and the ratio of the photocurrents is measured. Dowdall and Stretch ( 2 4 ) prefer a 12-volt tungsten lamp over the mercury vapor lamp as a n exciting source in a t w in-beam null point photomultiplier fluorometer for the analy-

sis of liquids. This lamp is reasonably rich in the longer ultraviolet rays, and many solutions are activated by rays just belon the visible region of the spectrum. Niki and Shirari (701 describe a n instrument for measuring the fluorescent spectra of solids where a photomultiplier tube is attached t o a spectroscope. The photomultiplier tube on the Beckman DU spectrophotometer makes this instrument much more adaptable t o the measurement of fluorescence and fluorescence spectra. A verj simple modification of this instrument which adapts it for eithei of these purposes is described by McL4nally (64). I n this case the lamp housing is replaced with one in which the lamp is removed and the solution may be inserted a t the focal point of the mirror. Another modification of the Beckman Model DT7 spectrophotometer places the fluorescent solution a t the exit slit and a photomultiplier tube a t the light entrance aperture (42). Brown and Narsh ( 1 4 ) use a modified Beckman Model D U t o measure the fluorescence of paper chromatograms. A strip transporting mechanism and a chart recorder are among the attachments used; with a 1P28 tube a concentration of 2.6 y of riboflavin can be detected. A combination of the Beckman D U and the Spekker fluorometer is described by Swann (83). Several new commercial fluorometers which employ photomultiplier tubes have been announced. One of these ( 6 4 )is designed t o measure the fluorescence of solids only and uses a fluorescent lamp as the activating source. -4nother commercial instrument is called a polyfluorimeter (3) and is designed t o measure the fluorescence of solutions or solids by transmission, reflection, 01 a t right angles. The light source for this fluorometer is a n H 100 A 4 lamp, and the photomultiplier tubes 931 A, 1P28, and 1P21 may be easily interchanged. -4third commercial fluorometer ( 7 6 ) , which also provides for a selection of photomultiplier tubes, is designed t o accommodate several different volumef of solutions. -4combination of two standard monochromators with a photomultiplier tube is offered by another apparatus com-

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A N A L Y TI C A L C H E M I S T R,Y

pany ( 6 6 ) for the measurement of fluorescent spectra and fluorescence excitation a s well a s for routine determinations. Bowman (11) and his associates have developed an instrument called a spectrophotofluorometer which is designed to excite and measure the fluorescence in either the ultraviolet or visible part of the spectrum. This instrument employs a B a u x h & Lomb monochromator t o analyze the excitation from a xenon arc source and a quartz prism spectrograph t o scan the fluorescence. T h e fluorescence intensity is measured with a 1P28 photomultiplier tube coupled t o a cathode ray oscilloscope. A review on fluorometry which includes general principles, instruments, solvents, and the effect of various conditions has been published by Indemans ( 5 2 ) . IIVORGANIC

Fluorescent x-ray analysis is an important phase of inorganic fluorescence analysis, but is not reviewed here because of the attention given this subject elsewhere (4, 62). The increasing application of inorganic fluorescence analysis is indicated by the inclusion of many fluorescence confirmatory tests in collegiate qualitative analysis. I n a book by Charlot ( l 7 ) , many fluorescence tests are given a n equal place with colorimetric reactions. A reference bibliography on fluorescence analysis with 180 titles has been compiled by Xonstantinova-Shlezinger (59). A very informative article on new fluorescence reagents for aluminum has been published by Holzbecher in Prague (47). This author has tested aluminum for fluorescence with compounds of the type of salicylaldehyde, 2-hydroxynaphthylaldehyde,and 18 of their derivatives. Salicylidene o-aminophenol a t a p H of 5 proved to be one of the best of these. The sensitivity is reported as 0.005 y of aluminum; the usual fluoride interference is noted. The fluorescence determination of aluminum and gallium with 8-quinolinol in chloroform has been in practice for some time, and now Collat and Rogers (19) have shown that these two elements can be determined in the same chloroform extract. The above authors show that the complexes of these two elements give a different response upon irradiation a t two different wave lengths and with the use of predetermined curves or equations they are able t o calculate the quantity of each element present in a mixture. This presents a new technique for fluorescence analysis and may have some interesting possibilities. I n this case the fluorescence spectra of the two complexes are essentially the same, but the fluorescence responses a t two activation points show a difference, I n expressing the fluorescence intensities these authors use a quinine equivalence, which is a good technique and will permit others t o check the experimental work. Another possibility for the determination of gallium or indium in the presence of aluminum is presented by Patrovsky (73). I n this method morin (2’,3,4’,5,7-pentahydroxy flavone) is used as an indicator for the titration with the disodium salt of ethylenediaminetetraacetic acid; the fluorescence of morin disappears a t the equivalence point of gallium and indium. If aluminum is present, fluoride ion is added to extinguish thefluorescence of the aluminum-morin complex. It i s of interest t o note t h a t morin is advocated as a colorimetric reagent for the determination of aluminum and 1 t o 1 ratio of morin t o aluminum is indicated (86). The authors did not have the equipment for a satisfactory fluorescence determination. Previous reports have indicated that the morin-aluminum composition of the f l u e rescent complex was 3 to 1. Toribara and Sherman (87) have concluded that morin is the most sensitive reagent for beryllium, which they have tried for the determination of micro quantities of beryllium in organic matter. A new reagent for beryllium, 2-(o-hydroxyphenyl) benzothiazole, has been suggested by Holzbecher (48). The reagent is 0.001M in 96% alcohol, and the test is performed in an acetate buffer a t a p H of 4.5. The fluorescence is bluish white, and several metals interfere, such as zinc, aluminum, antimony, and colored ions.

The use of the 8-quinolinol-aluminum complex as a qualitative reagent for fluoride has been tested by Harrigan (44)and is recommended over other methods. The test is performed on paper which has been dipped in a chloroform solution of the complex and dried. This coated paper held in hydrofluoric acid fumes becomes nonfluorescent. Bouman (10)has successfully used the aluminum-morin chelate for the determination of fluoride in concentrations of 0.02 y per nil. and advocates that the reaction be performed in 10% ethyl alcohol. An interesting fluorometric method for the determination of cadmium is reported by Evcim and Reber (25). The method involves the precipitation of cadmium with 2-(o-hydroxyphenyl) benzouazole. The precipitate is filtered and washed, the cadmium complex dissolved in glacial acetic acid, and the fluorescence measured. Concentrations of cadmium from 0.1 to 2.5 mg. per 50 ml. were used. This method is of particular interest because the reviewer believes that it illustrates the use of a fluorescent reagent which is restored, in glacial acetic acid, and the fluorescence of the original reagent rather than the complex is meaaured. Brodaks, Feigl, and Hecht (IS) have described a new application for the determination of gallium. Gallium is precipitated with a mixture of morin and cupferron and is extracted from a hydrochloric acid solution with chloroform. A 3 little a s 1 y of gallium in 6 ml. of chloroform is easily determined from a mixture containing 1000 times more aluminum, zinc, and iron, A qualitative fluorescence method for gallium with rhodamine B as the reagent has been developed by Onishi ( 7 3 ) . This reagent gives a n orange red fluorescence that will detect 0.1 y of gallium, and only a few elements interfere. Raju and Rao (80) have indicated that benzoin will serve as a fluorescence reagent for the detection of germanium. The reaction medium is alkaline 95% ethyl alcohol; the fluorescence is greenish yellow, and 10 y can be detected in 5 ml. of solution. As these conditions are identical with those used in a very delicate test for boron, the reviewer does not believe that this test for germanium will be very successful. I t does, however, suggest the similarity of germanium and boron, and tests for either element should be tried for the other. These same authors (79) show that resacetophenone in concentrated sulfuric or phosphoric acid gives a greenish gold fluorescence with germanium, whereas the boron fluorescence is blue. The limit of the test is 100 y of germanium. Feigl and Gentil ( S I ) have discovered that tin hydroxides form a blue-green fluorescence on paper with morin, which is stable in dilute acetic acid; aluminum, antimony, and zirconium give similar resulte. The method has been applied t o the determination of tin in allow, minerals, and mordanted silk (33). Fassel and Heidel ( 2 7 ) have determined the fluorescent spectra for terbium in terbium chloride solution and have shown that terbium can be determined quantitatively in amounts as low as 0.05 mg. in 10 ml. of solution. Calibration curves are given for terbium in the presence of other rare earths, and the effect of other ions is also shown. This method seems t o have distinct advantages over other methods for the determination of terbium and is unique in fluorescence determinations. in that no reagent is added and the intensity is measured at the narrow band fluorescence of the inorganic ion. The orange-red fluorescence of thallium v i t h rhodamine B will serve t o detect 0.03 y of thallium (36). Pollard and his associates (78) have published a series of six articles dealing with the analysis of inorganic ions by paper chromatography, and in this work a number of compounds have been investigated for separating and identifying metallic ions. For example, with the lanthanides it is shown that yttrium, lanthanum, and lutetium give the usual fluorescence with morin and 8-quinolinol but gadolinium fluoresces a deep brown with the latter and green with morin. Details for the determination of uranium in minerals contain-

V O L U M E 28, N O 4, A P R I L 1 9 5 6 ing less than 10 p.p.m. are given by Adams and AIaeck ( 2 ) . These authors use the sodium fluoride fusion and think it superior to the colorimetric method for small quantities. Zimmernian and Rabbitts (93) have reported further on the use of the uranyl fluoride method for the determination of uranium. Walton (89) has noted the quenching effect of plutonium and iron in the uranium determination. h highly selective fluorescence spot test for hydrazine has appeared in the Russian literature (61). The reagent is 1 gram of salicylaldehyde in 2 nil. of acetic acid and 100 ml. of water. This mixture is spotted on n drop of hydrazine solution on paper; an orange-yellow fluorescence results and 5 X 10-10 gram of hydrazine can be detected. Ammonia, azides, nitrate., and nitrites do not interfere. Charles and Freiser (16) in the fifth of a series of articles on organic analytical reagents have reported an interesting research on the chelates of 2-(-o-hydrouyphenyl)-benzoxazole, 2(o-hydroxyphenyl)-benzothiazole, and 2(o-hydrox~-pheny1)-benzothiazoline. Chelates of the first compound with lead fluoresce green, n-ith zinc blue, and with magnesium blue. The reagent is fluorescent, but chelates with the transition metals do not fluoresce. -1paper of general interest in trace analysis which gives a variety of possible fluorescence applications has been published by Irving and Rossotti ( 5 3 ) . These authors give a theoretical discuwion of sensitivity test ar!d tabulate the results of their experiments on the color and fluorescence of 33 cations n i t h 8quinolinol, 8-hydrouyquinazoline, and 5,8-dihydroxy-2,3-dimethyl quinoxaline. .4t present none of these reagents Peem superior t o 8-quinolinol, hut further tests are in order, ORGAVIC

The increasing popularity of fluorescence in organic analysis is indicated by the inclusion of a number of fluorescence reactions in two new books on methods of analysis of organic compounds. Feigl ( 2 8 ) has 29 applications of fluorescence and Pesez and Poirier (‘75) have 34. Both of these books are excellent references. Feigl and his associates (29) have discovered a sensitive and specific test that will detect 0.005 y of coumarin. \Then coumarin is dissolved in dlLali> the pyrone ring opens with the production of the alkali salt of o-hydroxycinnamic acid which becomes highly fluorescent after exposure t o ultraviolet light because of the formation of the trans configuration. Coumarin ie removed from impurities by vaporizing it a t 100’ C onto test paper moistened with alkali. -4rather extensive paper dealing with the separation of coumarin and its derivatives has been published by Berlingozzi and Fabbrini (9). Rhodamine B is recommended by Feigl and Gentil ( 3 0 ) as a fluorescence reagent t o detect enolizable polynitro compounds. These compounds give a red-violet salt with rhodamine B, which gives an orangered fluorescence in benzene. Stubner ( 8 2 )has shown that DDT can be detected under the microscope by its orange-iellon- or lemon-yellow fluorescence. h 58-page discussion on organic fluorescent and photochemical substances by Fujimori ( 3 8 ) viill be of general interest t o anyone dealing x i t h fluorescence. The article is in English, and the theory of fluorescence as related t o absorption and photosensitivity is discussed in some detail. Many absorption and fluorescence spectra curves are included and a great number of specific cases are given. For example, in one section the reaction of anthrone with formaldehyde, 613 cerol, etc., to produce six fluorescent compounds is illustrated by formulas and curves, and the effect of solvents is shown. Another section of the paper is devoted t o the “phototrophy of new photosensitive complexes between a fluorescent pigment and SH compounds.” The previous work of other authors on the separation of h5-drocarbon classes in light petroleum distillates by adsorption on silica gel and identification by fluoreqcent indicators has been extended by Harvey and Peareon ( $ 5 )

623 BIOLOGICAL

Fluorometric methods for the determination of steroids have been the subject of a number of excellent papers. Bates ( 6 ) , in a paper delivered before the Laurentian Hormone Conference, discussed spectrophotometric and fluorometric methods for the determination of estrogenic steroids. Goldzieher and Bodenchuk ( 4 1 ) studied the fluorescent reaction for 29 steroids. These authors list the wave length of peak intensity and the fluorescence intensity a t this and several other wave lengths. The influence of isomerism, unsaturation, and hydroxy groups is also included. Sweat ( 8 4 ) has made an extensive study of the fluorescence of corticosteroids in the presence of sulfuric acid and has also sho-xn the use of a silica niicrocolunin for chromatographic resolution of corital steroids (86). Abelson and Bondy ( 1 ) have described a simple fluorometric procedure for the analysis of ~~-3-ketosteroids. This deterinination is based on the reaction of the steroid with potassium tert-butoxide in tert-butyl alcohol and is applicable t o testosteronr, progesterone, and the biologically active adrenocorticol C, steroids. Braunsberg (12) and his associates have made an estrnsive study of the fluorescence of estrogens in phosphoric acid of and have found it possible t o measure, for example, 0.01 estradiol by the use of a photomultiplier fluorometer. Heusghem ( 4 6 )has shown that hydrogen peroxide destroys the fluorrscence of estrogens and that the residual fluorescence after the hydrogen peroxide treatment shbuld be subtracted from tlle original value. The use of fluorescence in the ultraviolet region of the spectra for the identification and determinat,ion of biological compounds hac; been developed by Udenfriend and coworkers (11, 88). These authors have developed instrumentation for activating and measuring fluorescence in the 250- t o 650-mp region of the spectra. Serotonin, for example, is activated a t 295 nip, and it: maximum fluorescence is measured at 330 mp. The authors give the activation wave length and fluorescent maximum for 20 compounds. Experiments indicate that the measurement of fluorescence in the ultraviolet n-ill be an important technique in analytical chemistry. Serotonin and other tryptamines have been shown by Jepson and Stevens (56) t o produce an intense blue-green fluorescence on paper when treated with 0.27, ninhydrin in acetone containing 10% by volume of glacial acetic acid. This test is sensitive to micromole per square centimeter. The spectral fluorescence and absorption of 3,4-benzopyrene has been the subject of a detailed study by Berg and Korden (8); the fluorescence spectrum changes from violet t o white-yellow with concentration and solvents. The fluorescence of pyridine nucleotides is affected i1y calcium and magnesium ions, and the depression of the fluorescence by these ions in the buffer becomes an important factor in the analysis of nucleotides ( 6 0 ) . The estimation of alkaline phosphates on blood serum has berm simplified by the use of fluorescence for the analysis of 2-naplitho1 (66). Freytag ( 3 6 )has developed a new fluorometric method for the determination of ascorbic acid and mercaptan compounds which is based on the reduction of 1,2-naphthaquinone-4-~~!fonate. iln article in Japanese, but with an English summary, also deals n-ith the determination of vitamin C ( 7 1 ) . Interesting factors affecting the fluorometric determination of ‘V-methyl nicotinamide have been found by Rosenthal ( 8 1 ) . Pretreatment of the samples mith alkaline or neutral peroxide may completely destroy the fluorescence and many elemen’? catalyze the formation of the fluorescent derivatives; iridium and cerium salts are especially effective. A fluorometric determination of cholic acid which is said t o be specific has been described by Pesez ( 7 4 ) . The procedure for the determination of gitoxin has been improved by the addition of ethyl alcohol t o the extraction media ( 3 7 ) . A simple method of separation of vitamins B1and Bz by paper chromatography and the use of fluorescence t o mark the cut and

624 estimate the material in each band is described by Giri and Balakrishnan (40). The fluorescent bands are cut from the paper and eluted for the final measurement of the fluorescence. A modified fluorometric procedure which is sensitive t o 5 per liter of adrenaline and noradrenaline is contained on a report from the Mayo clinic on the quantitative determination of these materials ( 6 6 ) The fluorescence and color intensity of antimony trichloride with bile acids may be used for the identification of a number of cholanic acids ( 1 5 ) . The fluorescence is specific for the position and number of hydroxyl or keto groups attached. S e u and Hagedorn ( 6 8 ) have shown that antimony trichloride is a better reagent than boric acid for the determination of the flavonols. These authors have tabulated the color and fluorescence of morin, quercetin, rutin, and others with this reagent. Horhammer and his associates have published a series of articles (43, 49-51) dealing with the separation, identification, and characteristics of the hydroxyflavones. The authors have studied in detail the reactions of boric acid and zirconyl ions with these compounds. Tables are given for the color and fluorescence of the zirconium and boron hydrolyflavone compleues, and evidence is presented to show that the hydrogen of the hydroxyl in the number 3 position is replaced by zirconium. Neu ( 6 8 ) has developed the procedure necessary for the use of boron as tetraphenyl diboronouide as a color and fluor2scence reagent for the hydroxyflavones and 8-quinolinol. Miller and Johnson ( 6 7 ) have presented a new fluorometric method for the determination of tryptophan, in which glucose reacts m ith tryptophan and the fluorescence of the resulting substance is measured. The detection of adrenaline and noradrenaline on paper chromatograms is simplified bv a reaction reported by Pitkanen ( 7 7 ) . Potassium ferricyanide is used as a n oxidant and the chromatogram is then treated with diniethylaminobenzaldehydr . adrenaline becomes dark blue and noradrenaline is changed t o a yellow compound m ith a strong I ellow fluorescence which fades as a blue color forms. The absorption spectra and fluorescence spectia of certain pigments of the porphyrin group have been published by Crow and Walker ( 2 2 ) in connection with a study of laboratory products isolated and purified by chromatographic and electrophoretic analysis. Becker and Kasha (6) give data on the fluorescent band of etioporphyrin I1 and its zinc complex, phthalocyanine and its magnesium complex, and pheophobide-a and chlorophyll a and b. Low temperatures narrow the fluorescent bands and give peak values which may be of use t o the analyst. The separation of porphyrins by paper chromatography and their detection by fluorescence have been described by Corwin and Orten (20). An .Imherlite column previously treated with sodium hydroxide has heen ueed to separate fluorescent impurities from vitamin Bl; the B1 is then eluted with hydrochloric acid ( 2 3 ) . -4s water solutions ofTen elute fluorescent materials from resins, a careful check on blanks must be made when this procedure is used. A fluorescent method is favored by Bencze ( 7 ) for the determination of small amounts of vitamin E. I n this method the tocopherol red is condensed with o-phenylenediamine in acetic arid, and the resulting phenazine derivative is adsorbed from a light petroleum solution on t o activated alumina. The compound ia eluted from the column Tyith a benzene-petroleum mixture and is taben up in a methanol-butanol mixture and its fluorescence is measured. Cahmann (18) has published excellent curves for the fluorescence spectrum of benzo-la] -pyrene and has shown the importance of both fluoreecence and absorption measurements in the determination of this compound in shale oil Lawrence and his associates have reported on some interesting cases of substances mhich are not fluorescent in water but become highly fluorescent when adsorbed on paper ( 3 4 )and have applied this principle to the determination of proteins (90).

ANALYTICAL CHEMISTRY For example, 1-anilinonaphthalene-8-sulfonic acid is nonfluorescent in aqueous solution but becomes brightly fluorescent when adsorbed on protein molecules. The fluorescence of blood plasma containing an excess of this dye was shoir-n to be a linear function of the albumin concentration. In the naphthalene and acridene series the introduction of an aniline group results in a compound nonfluorescent in aqueous solution but brightly fluorescent when adsorbed on a solid. RIavrodineanu (66) and his associates have shown that fluorescent materials on paper chromatograms separated from plant materials can be measured with a simple adaptation of a standard transmission density unit of the Photovolt Corp. photomultiplier photometer. The lower limit of measurement for indole%acetic acid was about 0.5 per spot. ?. review of the use of the photoelectric fluorometer and its biological applications is given hy Yagi and Tabata (92) with 24 references.

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LITER.4TURE CITED

(1) .4helson, D., Bondy, P. II., *ANAL. CHEM.25, 1865 (1953). Carey, J. B., Jr.. Bloch, H. S., J . Lab. Clin. X e d . 44,486(1954). Charles, R. G., Freiser, H., A n d . Chini. d c t a 1 1 , 1 (1954). Charlot, G., “Qualitative Inorganic Analysis,” Wiley, New P o r k , 1954. Cahmann. H. J., ANAL.CHEM.27, 1235 (1955). Collat, J. W., Rogers, L. B., Ibid., 27, 961 (1955). Corwin, L. AI., Orten. J. RI., Ibid., 26, 608 (1954). Crow, &I. O’L., Walker, A . , A p p l . Spectroscopy 8 , 57 (1954). Doede, C. M., Walker, C. A , Chem. Eng. 62, 160 (1955). Domange, L., Longuevalle, S., Pharm. Weekblad 90, 119 (1955). Dowdall, J. P., Stretch, H.. Analyst 79, 651 (1954). Evcim, S . K., Reber, L. A , , A 4 ~ .CHEM. t ~ . 26, 936 (1954). Farrand Optical Co.. Inc., New York, S . Y., Bull. 811 (1955). Fassel, V. A., Heidel, R. H., ANAL.CHEM.26, 1134 (1954).

a,,

Feigl, F., “Spot Tests Organic Applications,” Elsevier Press, Houston, Tex., 1954. Feigl, F.. Feigl, H. E., Goldstein, D., J . Am. Chon. Soc. 77,4162 (1955).

Feigl, F., Gentil, T’., ASAL. C m x 27, 432 (1955). Feigl, F., Gentil, V., Mikrochim. Acta 1954, 90. Feigl, F., Gentil, V., Goldstein, D., A n a l . Chim. Acta 9 , 393 (1953).

Feigl, F., Gentil, V., Goldstein, D.. Mikrochim. d c t a 1954, 93. Fildes, 3.E., Lawrence, D. J. R., Rees, V. H.. Biochem. J. (London) 56, XXXI (1954). Florida, C. D., Davey. C . S . ,J . Sci.I n s f r . 30, 409 (1953). Freytag, H., 2. anal. Cheni. 139, 263 (1953). Fruytier, J. F. -1.. Pinxteren. J. .I.c‘. van, Pharm. Weekblad 84, 99 (1954).

Fujimori, E., R e p f . I n s f . I d . Sei. mi?'. Tokyo 4 , 93 (1955). Galvanek. P.. Jr., LIorrison, T. J.. Jr., U. S. iltoniic Energy Comm. Rept. ACCO-47 (1951). Giri, K. V., Balakrishnan. S...ANAL. CHEM. 27, 1178 (1955). Goldaieher, J. IT., Bodenchuk, J. M.,Solan. P., Ibid., 26, 853 (1954).

Gornall, A. G., Kalant, H., Ibid., 27, 474 (1955). Hansel, R., Horhammer, L., Arch. Pharm. Berlin 287, 189 (1954).

Harrigan, AI. C., J . Assoc. Ofhc. A g r . Chemists 37, 381 (1954).

V O L U M E 28, N O . 4, A P R I L 1 9 5 6 (45) (46) (47) (48)

Harvey, P. G . , Pearson, R. l f . , Analyst 79, 158 (1954). Heusghem, C., S u t u r e 173, 1043 (1954). Holxbecher, Z., Chem. Lis& 47, 680, 1023 (1953). Holxbecher, Z., Collection Czechosloc. Chem. Communs. 20, 193

(1955). (49) Horhammer, L., Hansel, R., Arch. P h a r m . Berlin 285, 438 (1952). (50) Ibid.,286, 153, 425, 447 (1953). (51) Horhammer, L.. Xuller, K. II.,Ibid.,287, 310, 376 (1954). (52) Indemans, -4.W.M.,Cheni. Weekblad 50, 236 (1954). (53) Irving, H., Rossotti, H. S.,Analust 80. 245 (1955). Jarrell-Ash Co., Xewtonville, Mass., AIfr, Bull., 1954 JeDson. J . B.. Stevenb. B. J.. S u t u r e 172. 772 (1953).

Kacl, K., yecerek, B., T’ecerkova, J., Chundela. B., 6asopis LBkdfzi Ceskkch. 93, 621 (1954).

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