REVIEW OF FUNDAMENTAL DEVELOPMENTS IN ANALYSIS
I I 1
I
Fluorometric Analysis
I
’I
I
T
CHARLES E. WHITE Universify of Maryland, College Park,
review covers the 2-year period since the previous review (190) from approximately September 1955 to November 1957. Only a selected group of references are included. Danckwortt’s book on LuniineszenzAnalyse has been revised for its sixth edition by Eisenbrand (37). This edition contains 2072 references and 200 pnges of text more than the fifth edition. The references are classified by subject matter, and a table gives fluorescent tests for the elements listed according to the periodic table. Goto (67) and Tabata (175) have published reviews of fluorometry for inorganic and organic analysis, respectively. A good general discuEsion of fluorescence, especially as related to organic compounds, is givenby West (188, 189). In a chapter entitled “Fluorescence Techniques for the Enaymologist,” Laurence (114) gives a good discussion on fluorescence in general and a detailed treatment of the measurement and applications of polarized fluorescent light. Rowen (22) has given a short review on the use of fluorescence for industrial analysis. Interest in the determination of the intensity of ultraviolet light by chemical means has improved the uranyl oxalate actinometer. I n one case (152), excess ceric sulfate is added to both the irradiated oxalate solution and to a blank; the residual ceric ion is determined with a spectrophotometer. Another improvement (160) suggests the addition of excess permanganate and this excess is determined with the iodine liberated from potassium iodide. However, Hatchard and Parker (78) have shown that a potassium ferric oxalate actinometer is faster, more sensitive, and more accurate than the uranyl oxalate system. The ferrous ion produced by the action of light is determined with 1,lO-pheiianthroline as the indicator. Increased use of spectrofluorometers has prompted articles on errors in the determination of excitation and fluorescent maxima (24, 3.2, 80). Some factors to be noted are: the response of tlie phototube, the variation of the intensity of the exciting light with change of wave length, the overlap of the absorption and fluorescent emission curves, the calibration of the prism or grating, scattered light, and the relation of recorder HIS
Md.
response to speed of analyzer. An especially informative article by Parker and Earnes (145),detailing their experience with spectrofluorometers and filter fluorometers, shows that many published data are applicable to only one instrument and are thus misleading. The use of simple correction factors mould make this information more generally applicable. Articles by Stevens (171), Teal and U‘eber (178), and Milkey and Fletcher (127) contain good fundamental information on fluorescent analysis and are profitable reading for ITorkers in any phase of this subject. Sprince and Rowley (169) have recommended quinine sulfate from 0.005 to 0.17 per ml. in 0.1N sulfuric acid as a standard for excitation and fluorescent spectra. The authors give t h e x values a t 350 and 450 mp, respectively, for quinine sulfate. APPARATUS
The most encouraging news in fluorescent analysis for the past 10 years has been the commercial production of spectrofluorometers. The AmincoBoaman (5) and the Farrand (53) spectrofluorometers are double-grating monochromators with a xenon arc as light source and are furnished with or without recorders. These instrurnents determine the fluorescent spectra and excitation spectra of a solution in a few minutes, may be hand operated, and are useful for routine measurement of fluorescent intensity. With an additional monochromator, the Eeckman DK-1 spectrophotometer may also be used to record fluorescent spectra (12, 64). Other coinniercial spectrophotometers have been modified for the same purpose (10, 104). Fluorescent spectra may be measured with a simply modified spectrophotometer as the Beckrnan Model B (I%), a Unicam SP500 spectrophotometer (lob), or with a phototube attached t o a spectroscope (94) or spectrograph (21). Excitation spectra of papers may be determined with the use of the reflectance attachment on the Beckman spectrophotometers DU and DK-1 (11, 115). Research TTorkers the world over have contiiiued to devise fluorometers with special advantages for particular prob-
lems, for e:rample, in Russia (19, 80, 139), Japar (I%$), England (23, 69), and France (14). A new mercuric sulfide lamp, claimed to be unusually steady and laving an appreciable emission from 1250 to 360 mp, has been described a:; used in a fluorometer by Laurence (1 ( 3 ) . This lamp is now commercially amilable (116) in a photomultiplier jilter fluorometer designed for solutions, but which may be adapted for paper. A commercial instrument which is easily converted to an absorption meter is advertised (82). Another fluorometer is designed especially to measure the fluorescent intensity of solid samples such as cotton, paint, and cloth (61). Other instruments are reported from research workers for use with paper chroinatograms (110), for paper or solutions (8),and for uranium fusions (129). The measurement of fluorescence by the degree of blackness which it canses on a photographic film may have special applications (96). All chemical laboratories should have a dark box for the visual observation of fluorescence. Most of these have been homemade, but nom a commercial dark-box c:ibinet of approximately 1 cubic foot fcir the observation of mineral fluorescence is available (151). A new krypton arc’ lamp (193), which has a useful spectral range from 125 to 165 mp, is of interest for work in the vacuum range. lM croscopists applying fluorescence n-ill be interested in an article by Dangl (38) and in a Reichert equipment brochure which is available from a commercial firm in the United States (166) as well as in Europe. INORGANIC
The strucmture and characteristics of the fluorescent metal chelates of the o,o‘-dihydroxyazo compounds have been the subject of an intensive study by Freeman and Khite (58). A number of new compounds were prepared and the characteristics of the chelates with seven different metal ions were deternined. Formulas for the complexes were determined by both fluorescent and absorption methods and both gave the same results. The pH of the hydrolytic precipitation point and the p1-I of maximum fluorescence were VOL. 30, NO. 4, APRIL 1958
729
found to correspond. N,N’-Dimethylformamide mas used as a solvent in much of this work and was shown to be especially suitable in some cases. Holzbecher (85,86) has also prepared salicylaldehyde condensation products with a number of compounds and has determined the properties of the metal chelates of some of these with aluminum and zinc. Fluorescent spectra, absorption spectra, and fluorescent excitation spectra of a number of metal chelates which are in common use in the analysis of aluminum, beryllium, boron, and zirconium have been published by White, Hoffman, and Rfagee (192). Absorption and excitation spectra were found to be similar. The absorption maximum nearest the fluorescent emission was the strongest excitation point in most cases. The fluorescent spectrum band generally overlaps the absorption band a t the longest wave length. Michal (126) recommends quercetin (3,3’,4’,5,7-pentahydroxyflavone) as a general reagent for inorganic ions in chromatographic analysis. He has tabulated the results with this reagent for 27 metal ions. Fluorescent acid-base indicators have been the subject of reviews by Dangl (S9), Zharova and Zolotavin (196), and Kosheleva (109). The pH change characteristics have been determined for three new acid-base indicators (143). Beryllium. Motojima (130) recommends 8-hydroxyquinaldine for the fluorometric determination of micro quantities of beryllium. A straightline function is obtained for the fluorescence of a chloroform extract of this complex, containing from 0 to 40 y of beryllium. A sensitivity greater than quinizarin and equal to that of morin (2’,3,4’,5,7-peiitahydroxyflavone) is claimed. A 9-page report on the determination of beryllium ores by the morin method has been published by Riley (158).
Boron. A further study of benzoin as a reagent for boron has been the subject of two papers. Parker and Barnes (146) have shown that oxygen should be removed from this system and advocate an exciting light of 405 mp to decrease photodecomposition. White and Hoffman (191) show that a glycine buffer a t pH 12.8 is desirable and have determined the maximum emission of this complex a t 480 mp and the excitation peak a t 370 mp. Cyanide. Hanker, Gamson, and Klapper (77)have reported that cyanide may be determined with a combination of chloramine T and nicotinamide. Cyanogen chloride is formed and this cleaves the pyridine ring to form a highly fluorescent product. A precision to 2% in a range of 0.3 to 6 y of cyanide was obtained.
730
ANALYTICAL CHEMISTRY
Gallium and In,:lium. Ladenbauer and Slama (112) pr:ipose 5,7-dibromo-8quinolinol as a new reagent for gallium. The fluorescence oE the chloroform extract is measured or chromatographic techniques may be iised (167). Ladenbauer and her acsociates have also outlined procedureii where the commercial dyes, Solochrcine Red E.R.S. and are used in Solochronie Black \Y.E.F.A., a fluorometric method for gallium (111). Onishi and Sandell (144) have devised a method for the dctermination of gallium, where eithei, the absorption or fluorescence of the gallium-Rhodamine B complex is mewired after extraction with benzene frori hydrochloric acid solution. Both motin (44) and 8-quinolinol (73) are also in continued use for the analysis of gnllium. Peigl (65) states that gallium oupferronate shaken with chloroform arid morin provides a specific, sensitive ,,est for gallium. A critical comparison of 8-quinolinol and 8-hydroxyquinaldine (88) shows that interferences are diminished with the use of the former in benzene. The optimum pH extraction values.with benzene mere 5.1 to 8.2 and 7.0 to 8.5, respectively. Samarium and 3Curopium. Peattie and Rogers h a w made a detailed study of the fluoreiicence of samarium in calcium tungstai,i: (147) and calcium sulfate (148); sam:Lrium(III) is determined by its inclusion in the dried product of a calcium tungstate slurry, and it is coprecipitated with calcium sulfate and partly reduced by electrons from a Van de Graff accel,.rator. Under similar condition of eleztron bombardment, europium can also :le detected. Dieke, Hall, and Leopold (42, 43) have determined the fluorescc!nt lifetimes of some of the rare earth salts. Mercury, Thall:iim, Thorium, and Tin. Stolyarov l:l78) describes a n improvement on tlie fluorescent detection of thallium. A drop of a thallium solution is mi red on a microscope slide with ammonium iodide, is allowed to dry slow y, and is examined under the microscope while irradiated with light of 313 mp. The limiting concentration is 1 part in 10 million. This author also d1:scribes methods for the determination of mercury based on the fluorescence of mercurous chloride, bromide, and bromate. Milkey and Fletcher (127) halt made a thorough study of the fluorescence of the thoriummorin complex and have indicated that the fluorescent metliod with this reagent is slightly less sensi ;ive than the absorption method. Tin(:[V) has been shown (34) to give a fluortrscence with flavonol similar to that of xirconium with this reagent. The method is simple and is sensitive to 0.02 y per ml. The optimum conditions for the determination of tin(I1) with 7-a:nino-3-nitro-l-naphthalenesulfonic acid. have been deter-
mined (6) and the interferences have been noted. Uranium. The fusion technique for the fluorometric determination of uranium is improved if 2% lithium fluoride with 98% sodium fluoride is used in place of the carbonate flux (28). This mixture melts a t 900” C. and gives greater sensitivity than the carbonate. Rapid control methods for uranium determination in ores have been described with details by ICaufman (99). Adler (1) has described the bead test for uranium as ideal for field use with simple equipment. Construction details for a new sensitive instrument which measures the fluorescence of uranium plaques are described by Byrne (26). Stevens and his associates (172) have constructed a rotary unit for the efficient fusion of uranium samples. Thatcher and Barker (179) have found the conditions necessary for determining small quantities of uranium in natural waters and described an electric fusion apparatus. Grotta (72) shows that the life of uranium standards may be improved if they are allowed to remain in the original fusion dish and are coated with a resin. Uranium in relatively large amounts may be determined quickly within 15% by visual comparison; a fluorometer is also described (71). Solution methods for the determination of uranium are not popular, but the sulfuric acid method has been improved (13) and morin may also be used (181). Tungsten and Zirconium. A technique to avoid interferences in the fluorometric determination of zirconium with morin has been devised (65). The fluorescence is measured in 2N hydrochloric acid before and after the addition of (ethylenedinitri1o)tetraacetic acid. This substance removes the zirconium from the morin complex and destroys the fluorescence. The predominant species has been determined as having a zirconium-morin ratio of 1 to 2, but there is probably some 1 to 1 present. I n another method which uses morin (3),the interference of certain ions is removed by reduction with zinc powder. Morin may also be used to detect zirconium on paper chromatograms (4) Tungsten may be determined by measurement of the decrease in fluorescence which its ions cause in Rhodamine B solutions (158). Chemiluminescence. Cheniiluminescence is not properly classified under fluorescence but, because the measuring equipment is similar, limited references are given here. Siloxene will serve as a luminescent indicator in the titration of lead with chromate (102). The luminescence of 5-amino-2,3-dihydro1,4 - phthalazinedione (3 - aminophthalhydrazide, luminol) is affected by oxidizing agents as peroxides andhypochlorites and by catalysts as hemin, cupramI
monium salts, and ferricyanides. ICIethods with luniinol as an indicator are possible for the determination of each of these materials (136,163). New chemiluminescent indicators for acid-base titrations such as 2,4,5-triphenyliniidazole (lophine) (51) and N,N'-diallylbiacridinium compounds (12G) have been developed. Fluorescent X-Ray. Fluorescence in the x-ray region is a specialized branch of fluorescent analysis. Only two review references are recorded here (110, 162). BlOLO GlCAL
A spectral fluorescent study of about 55 compounds of biological interest has been made by Duggan and his associates (47). Values are listed for the activation maximum, the fluorescent maximum, the pH, and the sensitivity, as determined by the instrument used. Generalizations as to structural requirements for fluorescence in solution are also presented. Udenfriend and his associates (183) have reported on a similar study of 53 drugs. Tlie same types of data as indicated above are given for these substances. The fluorescent intensity of about 100 powdered drugs has been determined by the photographic film method (176). The fluorometric determination of adrenaline and noradrenaline in plasma and urine has been investigated extensively by Weil-Nalherbe and Bone (186, 187) and by Euler and Floding (52). Other papers on the determination of these substances in plasma (87, 157, l84), and in urine (70, 140) have appeared. Because of the possibility of forming more than one fluorescent product, controlled conditions in this determination are important (25). The fluorescence of ethylenediamine derivatives of adrenaline and noradrenaline is discussed by Ahngan and Mason (120). Errors in previous work are noted and the fluorescent spectra are included. These authors state that aluminatreated acetic acid gives a more intense fluorescence with these compounds than untreated acid. Potassium peroxydisulfate may be used to oxidize adrenaline and noradrenaline in alkaline solutions (121). An especially informative article on the ultraviolet fluorescence of aromatic amino acids has been presented by Teal and Weber (178). Apparatus is described for the determination of the excitation and fluorescent spectra and fluorescent spectra are given for tyrosine, tryptophan, and phenylalanine. Quantum yields are calculated and the fluorescent efficiency values are found to differ from those of Shore and Pardee (165). These latter authors have also described the determination of amino acids on paper by fluorescent derivatives
(164). An intensive study on indoxyls (5hydroxyifidoles) and related compounds has been published by Sprince, Rowley, and Jameson (170). Data on the fluorescent spectra and activation maxima are given for 16 compounds. Calculations based 011 Planck's energy equation shonr some relationsliipsmorthy of note. The paper chromatographic separations of 3-indoleacetic acid (heteroauxin) and certain fluorescent compounds related to coumarin are discussed by Pavillard and Beauchamp (146). A rapid, simple, spectrophotofluorometric method for the determination of tryptophan and tyrosine in protein hydrolyxates aiid of tryptophan in plasma has been described by Duggan and Udenfriend (48). The fluorescence of a reaction product of glucose and tryptophan may be used for the determination of micro amounts of tryptophan (128). A method for the fluorometric determination of heteroauxin in plants has been devised by Rakilin and Povolotskaya (155). The plant nisterial is extracted with an alcohol-ether mixture and the fluorescence of the extract is measured. Further treatment is used to determine the bound heteroauxin. The fluorescent spectra of 3,4,9,10dibenzopyrene in cyclohexane and its chromatographic separation from 3,4benzopyrene have been determined by Muel, Hubert-Habart, and Buu-HoI (132). The fluorescent metabolites of 3,4-benzopyrene have been fractionated from bile by paper chromatography for the fluorometric determination (SO). Hirshberg (83) has determined the fluorescent spectra a t liquid air temperatures of 1,2-benzanthracenes and methylbenzo [c]phenanthrenes, and he shows that changes in the fluorescent spectra are evidence of the position of the methyl group on these compounds. The chemical evaluation of Rauwoljia serpentina alkaloids has claimed the attention of a number of investigators. Dechene (41) shows that the fluorescent, intensity of a reserpine solution is increased 10-fold after oxidation with hydrogen peroxide. Gordon and Campbell (66) use ceric sulfate in the determination of canescine (11-demethoxyreserpine) and state that the fluorescence is increased 10-fold with this reagent, also (fluorescence maximum a t 360 mp and activation a t 280 mp). Reserpine after separation by electrophoresis may be determined in solution (76) or on paper after being intensified with hydrogen peroxide (105) or sodium nitroprusside (27). Several methods are available to remove impurities likely to interfere in the fluorescent method (163). Gyenes and his associates (74) have described the fluorometric determination of the ergot alkaloids by means of the Pulfrich fluorometer and use this to indicate the monohydrogenated alka-
loid content of hydrogenated ergotoxinetype alkaloills (75). A theoretical discussion, which is interesting and geneial, on the absorption and fluorescence of steroids, has been presented by Arrhenius ( 7 ) . The fluorescent Epectra in sodium hydroxide for estrone, 17-p-estradiol, and estradiol have been cleterniined by Nakao and Aizawa (187). Light a t 365 mp was used for excitation and all of these substances had their maximurn emission a t 480 mp. The authors have also described mic -oseparation of these estrogens by cliromatography on an alumins column (1%'). A paper chromatographic separation of these estrogens by a unidimensional ascending system has been found efficient by Puck (154). A method applicable to routine clinical testing of estrogens in urine is described by Rlasek m d Janda (122). I n work with bovin 3 urine, biological assays indicate that only one quarter of the observed fluorwcence is due to biologically active estrogens (168). I n sulfuric acid solutions, the fluorescence of some estrogens decrease with time, whereas the background fluorcscence does not show this time factor. This difference may be used to correct the apparent estrogen content for the fluorescence due to impurities (174). Kalant (07) uses an ethylene chloride extract of the neutralized sulfuric acid solution for paper chromatogrephy of steroids. The microanalysis of A4-3-oxosteroids by paper chromatogr:,phy and by development on the paper with sodium hydroxide is described by Ayres, Simpson, and Tait (9). The intensity of the fluorescence is proportional to the concentration if the amount of the steroid on the spot is less than 4 'I. Albers and Lowry (2) determined 0.1 to 10 y of cholesterol in animal tissue by measurement of the fluorescence of a sulfuric acid solution after extraction with ethyl alcohol. High sensitivity is obtained mi thout sacrifice of precision. Udenfriend and his associates have found that serotonin in 3N hydrochloric acid with an activation wave length of 295 mp gives a maximum emission a t 550 mp (182.t and have applied this in an assay of serotonin extracted from brain
(18). A new spot test for steroids in which their fluorescence is developed by p-toluenesulfonic acid appears simple and shows a more intense fluorescence than by sulfuric acid development. The steroid is placed in a spot plate, overlaid with crystals of the acid, and heated to a melt a t 110" to 120" C. A table is presented M hich gives colors and the fluorescent color of about 30 steroids with this technique (50). A study of the effect of various compounds on the fluorescence of riboflavine shows that phenolic, hydroxyl, amino, or other groups with a strong solutionVOL. 30, NO. 4, APRIL 1958
* 731
increasing activity have a quenching effect. Cyclic compounds without these groups have no effect. Borax combines and also has a quenching effect (161). Alcthods and solvent systems for chromatography of flavines and flavine nucleotides have been described in some detail and seiniquantitative results were obtained by photography of the flavines under ultraviolet light (105). I
