V O L U M E 22, NO. 1, J A N U A R Y 1 9 5 0 In extremely dilute solutions the color change is from faint yellow (acid) to pink (alkaline) for I and IV and from faint yellow to ambar for I1 and 111. I n strongly alkaline medium the color fadrs, but the sluggishness of this color change renders it useless f o r indicator purposcs. The properties of the compounds are summarized in Table TI. The mechanism of the color change is given by:
69
Colorless Stock solutions (0.1%) in alcohol are prepared. It is not stated whether any of the indicat.ors have advantages over phenol red or other Clark and Lubs indicators which have about the same color change interval. LITERATURE CITED
Faint yellom-
+
OH-
+
Pink or amtwt
(1) Belcher, It.. Anal. Chim. Acta, 3, 578 (1949) (2) Raykhinstein, 2. G., and Kocherigina. T. V., J . Anal. Chem. (Russia), 2, 173 (1947). (3) Schulek, E., Z. anal. Chem., 102, 111 (1935). (4) Schulek, E.. and Rozsa. P., Ibid., 115, 185 (1939). (5) Schulek, E., and Somogyi, Z., Ibid., 128, 398 (1948). (6) Smith, G. F., and Brandt, IV. M., A h a CHEM., ~. 21, 948 (1949).
(7) Stewart, R., and Clark, R. H., Can. J . Research, B26,7 (1948). (8) Usel. F. L., Casopis Ceskdho LQkdrnictva, 15, 143 (1935). RECEIVEDS o v e m b e r 14, 1949.
FLUOROMETRIC ANALYSIS CHARLES E. WHITE, University of M a r y h n d , College Park, M d .
A
LTHOUGH this review is concerned primarily with the application of fluorescence to analytical problems, it is worth while t o call attention to a book on fluorescence and phosphorescence by Pringsheim ( 5 7 ) . This is a much enlarged and revised edition of the author’s previous book on luminescence. The analyst \Till find of particular interest the discussion on the theory of fluorescence, and the sections on the fluorescence of organic compounds and the luminescence of pure inorganic compounds. The Russian Academy of Science has published a book on luminescence analysis, edited by Konstantinova-Shlezinger (39). Because this is entirely in Russian, it will find but little use in America until a translation appears. This same author has given a short review on fluorescence analysis (38). Feigl(22) devotes Chapter X I 1 of his book on the “Chemistry of Specific, Selective and Sensitive Reactions” to the analytical use of fluorescence effects. This is so brief that many applications are merely named and much recent work is not included. In a new book on “Methods of Quantitative Analysis,” Milton and K a t e r s ( 4 8 ) give a short discussion of fluorometric analysis with directions for the analysis of some few elements. Unfortunately, their references on this subject, except in one case, do not go beyond 1943. A critical revien. of fluorescence microscop\- has been given by Haniperl(29). APPARATUS
In a similar review last year (?I), the 360 B.L. phosphor lamps were mentioned as a new source of ultraviolet radiation for fluorometric work. These have now been improved t o give over twice the ultraviolet output of the earlier models and the maximum is now at about 3500 A,, whereas the former was a t 3600 A. Curves giving a comparison of these lamps have been published in a bulletin of the Sylvania corporation (69). Analysts using the Klett fluorometer will be interested in an article by Slater and Morel1 (67) in which are given many suggestions on the use of this instrument, especially with reference to its use in determining thiamine and riboflavin. The use of the Pfaltz and Bauer instrument has been smplified by a test tube adapter which has been described by Durst and Lewis ( 2 1 ) and Myers (60). In spite of the efforts of commercial manufacturers, many
analysts prefer to build their own fluorometers. Price ( 5 6 ) and others, in a report from the Atomic Energy Commission, describe an instrument which they have designed to determine uranium by the fluorescence of the uranium-sodium fluoride melt. Their present instrument is of simple design, using a photomultiplier tube. I t is sensitive to 10-11 gram of uranium and the authors indicate that a still more desirable instrument will be described in a forthcoming publication. A simply constructed photoelectric colorimeter and fluoromrter is described by XcGillivray ( 4 5 ) . For photographing the fluorescence spectrum, Scheminsky ( 6 4 ) uses a hand spectroscope and a Contax camera, and reports excellent results. Fluorescence microscopy has many applications and a simple device including types of filters for this purpose is described by Zamkov (72). INORGANIC APPLICATIOh S
An interesting technique for the identification of the cations by using paper chromatography in conjunction with fluorescence has been reported by Pollard ( 5 5 ) and his eo-workers. The cations are dissolved in a solvent consisting of water, butanol, acetic acid, and acetoacetic acid ester, or other appropriate mixtures. This is dropped on Whatman KO.1 filter paper, so as t o give spots about 1.3 em. in diameter and 4 em. apart. The paper is then sprayed with morin, oxine, etc., and observed under ultraviolet light. Twenty-four cations can he immediately detected by this procedure. The quantitative determination of oxygen (40)in quantities of 0.01 to 20 p.p.m. can be accomplished by permitting it t o oxidize leucofluorescein to fluorescein. Stable solutions of the reagent are prepared by reducing fluorescein with sodium amalgam and storing it under a light petroleum fraction. As little as 1 X lo-” mole of oxygen (33)can be detected by its effect on trypaflavin adsorbed on silica gel. Oxygen destroys the orange fluorescence of this compound and a greenish hue appears. Several references to the analysis of beryllium in rocks were reported in this review last year. Sandell (63) has made some revision of his method in which morin is used as the reagent and shows that as small an amount as 0.05 microgram of beryl3 p.p.m., lium can be identified. Granite containing as low was analyzed successfully.
=
70
ANALYTICAL CHEMISTRY
Rodden (60) has described a fluorometric method for uranium which detects as little as lo-” gram using the sodium fluoride flux technique. This same technique has been investigated by Price ( 5 6 ) and his co-workers, who have made an extensive study on the effect of inhibitors on the uranium fluorescence. Gibbs and Evans ( 2 5 ) describe a portable instrument for measuring the fluorescence in this procedure. Boutaric and Maraux (9) have studied the effect of inhibitors on uranium solutions. This is of obvious interest to analysts using the solution method. The rare earths (19) may be detected in coal ash in quantities which are too small for spectrographic analysis by mixing the ash with calcium oxide and subjecting it to cathode rays. The resulting luminescence is so bright that an exposure of only 5 to 12 minutes is necessary to photograph its spectra. In the mineralogical field Cannon and Murata (13) have shown t h a t the molybdenum content of scheelite can be rapidly determined by comparing the fluorescence of the sample with a standard. The fluorescence of mineral zircon is claimed by Foster ( $ 8 ) to have considerable practical significance in petrological problems. A new technique for observing the fluorescence of minerals with a microscope or spectroscope is described by Komovskil ( 3 7 ) . The luminescence of optical glass (11) under ultraviolet excitation provides a means of rapidly analyzing specimens for classification into commercial types. Sill and Peterson (66) have described a unique and useful qualitative method for thallium, which employs the bright blue fluorescence of the thallous ion, in sodium chloride solution, under the influence of short-wave ultraviolet light. The sensitivity is 1 in 50,000,000 and there are no ions that give a similar fluorescence under these conditions. APPLICATIONS IY BIOLOGICAL CHEIIISTRY
A general review of the biological applications of fluorescence analysis has been given by Radley (68). The fluorometric determination of adrenaline in the blood has been the subject of considerable study. Block ( 7 ) points out t h a t the blood contains an inhibitor for the fluorescence of adrenaline which can be removed by dialysis and is destroyed by prolonged boiling or by ultraviolet irradiation. Annersten (3) and his coworkers show that determinations made, using the dialysis method, give results closely corresponding to other methods. Pekkarinen (53) uses aluminum hydrolide to adsorb adrenaline before applying the reaction to produce the fluorescence, The fluorometric methods for adrenaline are discussed by Lund ( 4 4 ) . The quantitative fluorometric determination of estrone and estradiol has been developed by Jailer ( 3 2 ) , who shows a linear relationship bet\yeen the fluorophotometer readings and the concentration of these compounds in sulfuric acid solutions. Boscott ( 8 ) , by using suitable solvents with the phosphoric acid test for natural estrogens, has obtained characteristic color or fluorescent reactions for several of these and has developed a quantitative procedure for dienestrol. Mariani ( 4 6 ) uses the Pulfrich fluorophotometer for determining estrone in the range of 6 to 30 micrograms. The determination of folic acid by the fluorescence of its oxidation product has become an established procedure. A411frey( 1 ) and others show that interferences may be removed by adsorbing the oxidation product on Florisil and eluting with borax solution. Application of the procedure shows good agreement n i t h the microbiological method. Andreeva and Bukin ( 2 ) inJicate that for high protein materials an enzyme treatment is necessary t o liberate all of the folic acid. The estimation of nicotinamide in the presence of other nicotinic acid derivatives is accomplished by Chaudhuri and Kodicek (15) by treating the sample with cyanogen and permitting it to stand in an alkaline medium to form the fluorescent compound. Results are given for the analysis of various organs, wheat products, and yeasts. A method for the determination of
the 6-pyridone of X’-methylnicotinamide in urine has been reported by Rosen ( 6 1 )and others. The fluorescent spectra of 3,4-benxopyrene has been studied by Bergol’ts ( 6 ) and his associates as a means of identificatioir in the animal organism. This method has been used for detecting benzopyrene in the urine of mice after 0.25 mg. had been injected subcutaneously. The analysis of pamaquine in blood plasma is carried out in concentrated sulfuric acid solution. This serves for a range of 10 to 500 micrograms of S.X.-13276 and S.N.-3291 and is applicable even when these substances are mixed with quinine (31). Fluorometric techniques in the identification and determination of porphyrins are in general use. Kliewe (34) shows that the intensity of the fluorescence of these compounds is greater in phosphoric acid than in hydrochloric. Comfort (18) has e\amined over 3000 molluscan shells, some of which were over 100 years old, and has shown that the red fluorescence of porphyrin is just as strong in these as in fresh material. Riboflavin decomposition products have been studied by paper partition chromatography and subsequently examined under ultraviolet light by Hais and Pec&kov&( 2 8 ) . The characteristic fluorescence of lumiflavin and lumichrome was observed in sddition t o other new spots. De Ritter (20) and others give a critical review of the methods for the determination of riboflavin in urine. This determination is also described by Wang ( 7 0 ) and his associates. The determination of riboflavin in blood has been dealt with by several authors (12, 26, 2 7 ) ; in some cases it is recommended that the total fluorescence and also that remaining after the addition of hydrochloric acid be determined. The riboflavin fluorescence is quenched by the acid and is determined by difference. Bessey (6) and his associates have devised a method for determining the nucleotides of riboflavin in biological material and give comparison data for the fluorometric and enzymatic methods. The former is much simpler and equallbas accurate as the latter. By use of a sensitive fluorometer Kodicek and Wang (36) have determined the riboflavin content of various meats and vegetables without concentratian by adsorption. Salicylates in the blood may be determined by the strong blue fluorescence in alkaline solution. Proteins are removed by precipitation with tungstic acid and Saltzman (62) has obtained excellent recovery of salicylate over a range of 0.02 to 0.5 mg. The fluorometric determination of thiamine by the tentative A.O.A.C. method has been the subject of a collaborative study and found to give good results. It is recommended that the method be made official (35). Patrick and Wright (52) recommend the use of mercuric oxide as an oxidant in the analysis of pharmaceutical products for thiamine. Their method is apparently more rapid and gives more accurate results than where ferricyanide is used. Ridyard (59) gives a number of refinements that experience has shown to be of utmost importance for the attainment of speed and accuracy in the determination of thiamine in wheat products. hlawson and Thompson ( 4 7 ) shoty how to avoid the interference of nicotinamide methyl chloride in urine analysis and Bowman (10) has outlined a method for determining thiamine in potatoes. Nen-man (51) and his associates have applied the sodium fluoride fusion method to the determination of uranium in biological materials. As little as 0.005 microgram of uranium per gram of tissue may be detected. The use of fluorescent dyes as tracers in biology and medicine has been reviewed by Lange (41). This article is written in a popular style and covers the use of fluorescence in ophthalmology, blood circulation, etc. Analysts interested in the determination of chlorophyll will find much pertinent information in an article by Livingston ( 4 3 ) . He shows that chlorophyll dissolved in pure hydrocarbons is practically nonfluorescent. The addition of 0.01 yo more water, alcohol, or amines raises the intensity of the fluorescence to
V O L U M E 2 2 , NO. 1, J A N U A R Y . 1 9 5 0 the same as when alcohol is used as a solvent. The absorption spectrum of chlorophyll in these solvents is also given. FLUORESCEYCE IN ORGANIC ANALYSIS
-1 quantitative determination of citric acid, based upon its transformation into the highly fluorescent compound ammonium citrazinate, has been developed by Leininger and Katz (42). From 10 to i 5 micrograms of citric acid give good results even in the presence of tartaric and malic acids. The application to the analysis of citrus fruit juices is described. Glycerol in dilute aqueous solutions can be easily identified by a reaction with 2,7-naphthalenediol in concentrated sulfuric acid. The aldehyde acrolein, formed from the glycerol, condenses. with the reagent and forms a yellow to reddish yellow solution which a h o m a strong green fluorescence (24). Purine and pyrimidine derivatives can be identified on chromatogram spot test paper (30)if excited with wave nlotions in the order of 2500 A. The reaction of malic acid and succinic acid with resorcinol in concentrated sulfuric acid has been used by Barr (4)t o determine these acids. V h e n the solution is made alkaline it becomes highly fluorescent. Phillips ( 5 4 ) has described a method of identifying a-amino acids and dipeptides by examination of chromatograms under ultraviolet light. The fluorescent spectra of many polvcyclic aromatic hydrocarbons in solution have been determined by Schoental and Scott ( 6 5 ) . A correlation is shown between the position of the spectrum and the number of quinoid rings in the structure. AY4LYSIS Q F PHARMACEUTIC4L PRODUCTS
Chase and P r a t t (14)have developed a system of identification for powdered vegetable drugs by means of their fluorescence directly or after treatment with sodium hydrouide. illcoho1 extracts were also used. One hundred and fifty-one vegetable drugs were studied and useful data are tabulated. The fluorometric determination of the ketotetrahydropyridenes in urine has been improved by D e Ritter (19) and his associates, so t h a t good results are obtained a t excretion levels as lo^ as 1 mg. per day. Antrycide, a new trypanciodal drug, may be determined in blood plasma b y its reaction with eosin, wherein the eosin salt of the alkaloid is estracted n+th butanol and chloroform and measured in a fluorometer (68). .$ fluorescence study of Bonitreager reaction for anthraquinone drugs has been made by Christensen and Abdel-Latif ( 17 ) . T h e authors conclude that the color of the fluorescence is specific for each of the drugs containing emodin and can be used in their identification. In order to increase the ease of observation, these same authors advocate the use of capillary tubes as containers for the solutions ( f 6 ) . LITERATURE CITED
Allfrey, V.. Teply, L. J., Geffen, C., and King, C. G., J. Biol. Chem., 178, 465 (1949). Andreeva. N. A , , and Bukin, V. N., Doklady A k u d . r a u k S.S. S.R., 64, 95 (1949). Annersten, S . , Gronwall, A . , and Koiw, E., ‘l’ature, 163, 136 (1949). Barr, C. G., Plant Phvsiol, 23, 443 (1945). Bergol’ts, V. hf., Il’ina. A. A, and Baailevich, V. V., B w k h i m i y a , 14, 20 (1949). Bessey, 0 . A., Lowry, 0. H., and Love, R. H.. J . Biol. Chem., 180, 755 (1949). Blork, TV.. H e h . Phvsiol. et Pharmacol. Acta, 6 , 122 (1948). Roscott, R. J., S a t i r e , 162, 577 (1948). Boutaric, M.A , , and Maraux. C., Bull. S O C . chim.France, 1948, 9.52. Bon-man, J., Intern. Rev. Vitamin Research, 19, 386 (1948). Brumherg, E. M.. Sverdlov, 2. 11.. and Timofeera, T. V , , Bull. acad. sci. [J.R.S.S., Sir. p h y s . . 13, 242 (1949).
71 Burch, H. B., Bessey, 0. A . , and Lowry, 0. H., J . Bid. Chem., 175, 457 (1948). Cannon, R. S., and Murata, K. J., U. S. Patent 2,346,661(1944). Chase, C. R., and Pratt, R., J . 4 m . Pharm. Assoc., 38, 324 (1949). Chaudhuri, D. K., and Kodicek, E.. Biochem. J . , 44, 343 (1949). Christensen, B. V., and Abdel-Latif, I. A , . J . Am. Pharm. Assoc., 38, 490 (1949). Ibid., 38, 487 (1949). Comfort. A,. Biochem. J . . 44. 111 11949). De Ritter, E., Jahns, F. W.,’ and Rubins, S. H., J . Am. Pharm. Assoc., 38, 319 (1949). De Ritter, E., Moore, M . E., Hirschberg, E., and Rubin, S. H., J . Bid. Chem., 175, 883 (1948). Durst, R. L., and Lewis, J. B., ANAL. CHEY.,20, 7 8 2 (1948). Feigl, Fritz, t r . by Oesper, R. E., “Chemistry of Specific, Selective and Sensitive Reactions.” New I’ork. Academic Press. 1949. Foster, W. R., Am. Mineral., 33, 724 (1948). Furst, K., Mikrochim. Acta., 34, 25 (1948). Gibbs, T. R. P., and Evans, H. T., Science, 105,72 (1947). Gourevitch, A,, Bull. soc. chim. b i d , 30, 711 (1948). Graziani, G., and Giordano, M., Bull. soc. ital. b i d , 24, 685 (1948). Hais. I. M..and Pecbkovh. L.. ‘Vature. 163.. 768 - (1949). ~, Hamperl, H.,Mikroscopie,’2, 152 (1947). Holiday, E. R., and Johnson, E. A , . S a t u r e , 163, 216 (1949). Irvin, J. L., and Irvin, E. M., J . B i d . Chem., 174, 389 (1948). J . Chem. Endocrinol., 8, 564 (1948). Jailer, J. W., Kautsky, H., and Muller, G. O., 2. Naturforsch., 2A, 167 (1947). Kliewe, H., 2. ges. inn. Med., 3, 543 (1948). Kline, 0. L., J . Assoc. Ofic. Agr. Chemists, 31, 455 (1948). Kodicek, E., and Wang, Y . L., Biochem. J., 44, 310 (1949). Komovskii, G. F., Bull. acad. sci. C . R . S . S . , Sir. p ‘ y s . , 13, 248 (1949). Konstantinova-Shlezinger, M .A., Ibid., 13, 237 (1949). Konstantinova-Shlezinger, M. d.,“Lyuminestsentnyi hnaliz,” Moscow, ilcad. Sei. S.S.S.R.,1948. Kulherg, L., and Matviev, L., J . Gen. Chem. C.S.S.R., 17, 457 (1947). Lange, K., J . Electrochem. SOC.,95, No. 6 , 131C (1949). Leininger, E., and Kate, S.,AXAL.CHEM., 21, 811 (1949). Livingston, R., Watson, W.F., and hIcArdle, J., J . Am. Chem. SOC.,71, 1542 (1949). Lund, A , , Acta Pharmacol. Toxicol., 5 , 75 (1949). McGillivray, T T . d.,Sew Zealand J . Sci. Technol., 29B, 317 (1948). bfariani, A , and Tentori, L., Rer~d.ist. super. sanitb, 11, 1176 (1948). Mawson, E. H., and Thompson, S. Y., Biochem. J . , 43, 2 (1948). Milton, R. F., and Waters, W-. A4.. “lMetho,ds of Quantitative Analysis,” London, Edward Arnold Co , 1949. Mukherjee, B., A-ature, 163, 402 (1949). Myers, C. S., Comm. Fisheries Rev., 11, 8 (1949). Newman, W.F., Fleming, R. TV., Carlson, A . B., and Glover, N., J . Biol.Chem., 173, 41 (1948). Patrick, R., and Wright, J. F. H., Analyst, 74, 303 (1949). Acta Physiol. Scand., 16, Suppl., 54 (1948). Pekkarinen, .I., Phillips, D. M. P., .L7ature, 161, 53 (1948). and Elheih, I. I. M.,Ibid., Pollard, F. H., McOmie, J. F. W., 163, 292 (1949). Price, G. R., Ferretti. R.J., and Schwartz, S., U. 9. Atomic Energy Comm., Bull. ACED 2282. Pringsheim, Peter, “Fluorescence and Phosphorescence,” New York, Interscience Publishers, 1949. Radley, J. A., M j g . Chemist, 19, 193 (1948). Ridyard, H. N., Analyst, 74, 18 (1949). Rodden, C. J., AN.~L. CHEM.,21, 333 (1949). Rosen, F., Perlaweig, TV. A , , and Leder, I. G., J. B i d . Chem., 179, 158 (1949). Saltaman, A., Ibid., 174, 399 (1948). Sandell, E. B., A n a l . Chim. Acta, 3, 89 (1949). Scheminsky, F., Spectrochim. Acta. 3, 191 (1948). Schoental, R., and Scott, E. J. Y . ,J . Chem. Soc., 1949,1683. Sill, C. W., and Peterson, H. E., ANAL.CHEM.,21, 1266 (1949). Slater, E. C., and Morell, D. B., Australian Chem. I n s t . J . Proc., 15, 221 (1948). Spinks, .I.,S u t u r e , 163, 954 (1949). Sylvania Electric Products, Boston, Eng. Bull. 0-72 (November 1948). \Tang, Y. L., Ting, K. S., Leh, H., Chao, J., and Hu, Y . H., Sci. Technol. China. 1, 33 (1948). White. C. E., A I ~ . 4CHEM., ~.. 21, 104 (1949). Zanikov, V. h.,Mikrobiologiya, 17, 400 (1948). j~
RECEIVED Sovember 10. 1949.
