(228D) Yanagisawa, M., Suzuki M., Takeuchi, T., Anal. Chim. Ada, d Z , 386 (1970). ,-_. -,-
(229D) Yudelevich, I. G . , Vall, G. A., Torgov V. G., Korda, T. M., Zh. Anal. khim., 25, 870 (1970); J . Anal. Chem. USSR,25,752 (1970). Atomic Fluorescence Spectrometry (1E) Aggett, ,J., West, T. S., Anal. Chim. Acta, Ac 55, 349 (1971). (2E) (2E Alder, J. F., West, T. S., ibid., 51, 365 (1970). (3E) Amos, M. D., Bennett, P. A., Brodie, K. G., Lung, P. W. Y., Matousek, J. P., ANAL.CHEM.,43, 211 (1971). (4E) Anderaon, R. G., Maines, I. S., West, T. S.. Anal. Chim. Acta,. 51,. 355 (1971’). (5E) Bailey, B. W., ibid. 54, 537 (1971). (6E) Belyaev, Yu. I., daryakin, A. V., Pchelintsev, A. M., J. Anal. Chem. USSR, 25,735 (1970). (7E) Belyaev, Yu. I., Pchelintsev, A. M., ibid., p 1799. (8E) Zbid., p 1922. (9E) Black, M. S., Glenn, T. H., Bratzel, M. P., Winefordner, J. D., ANAL. CHEM.,43, 1769 (1971). (10E) Bratzel, M. P., Dagnall, R. M., Winefordner, J. D., Anal. Chim. Acta, 48,197 (1969). (11E) Ibid., 52, 157 (1970). (12E) Bratzel, M. P., Dagnall, R. M., Winefordner, J. D., Appl. Spectrosc., 24,518 (1970). (13E) Browner, R . F., Dagnall, R. M., West, T. S., Anal. Chzm. Acta, 50, 375 (1970).
(14E) Cotton, D . H., Jenkins, D. K., S ectrochim. Acta, 25B, 283 (1970). (158) cresser, M. S., West. T. S., Anal. Chim. Acta, 50,517 (1970). (16E) Ibid., 51, 530 (1970). (17E) Cresser, M. S., West, T. S., Spectrochim. Acta, 25B, 61 (1970). (18E) Cresser, M. S., West. T. S., Spectrosc. Letters 2 , Q (1969): (19E) Dagnad, R. M. Kirkbright, G. F., West, T. S., Wood, k.,Analyst, 95, 425 (1970). (20E) Dagnall, R. M., Kirkbright, G. F., West. T. S.. Wood., R.., ANAL. CHEM.. 42,1029 (1970). (21E) Zbid., 43, 1765 (1971). (22E) Dagnall, R. M., Taylor, M. R. G., West, T. S., Lab. Prmtict 20,209 (1971). (23E) Denton, M. B., Mafmstadt, H. V., Appl. Phys. Letters, 18, 485 (1971). (24E) Ebdon, L., Kirkbright, G. F., West. T.S., Anal. Chim. Acta, 47, 563 (1969). (253) Ebdon, L., Kirkbright, G. F., West, T. S., Talanta, 17, 965 (1970). (26E) Elser, R. C., Winefordner, J. D., Appl. Spectrosc., 25, 345 (1971). (27E) Fraser, L. M., Winefordner, J. D., ANAL.CHEM.,43, 1693 (1971). (28E) Fulton, A., Thompson, K. C., West, T. S., Anal. Chim. Acta, 51, 373 (1970). (29E) Hobbs, R. S., Kirkbright, G. F., West, T. S Talanta, 18,859 (1971). (30E) Kirkbilght, G. F., Analyst, 96, 1146 (1971). (31E) Kirkbright, G. F., Rao, A. P., West, T. S., Anal. Lett., 2, 465 (1969). (32E) Larkins. E’. L.. Smctrochim. Acta, 268, 477 (1971). I
.
’
(33E) Larkins, P. L., Willis, J. B., ibid., 491. ((34E) 3 G J Lowe, R. M., ibid., p 201. (35E) Marshall, G . B., West, T. S., Anal. Chim. Acta, 51,179 (1970). (36E) Martin, T. L.. i36E) L., Hamm. Hamm, F. M.. M.,’ Zeeman, P. B., ibid.,‘53, ibid., 53, 437 (1971). . Zkman, (37E) Miller, R. L., Fraser, L. M., Winefordner, J. D., Appl. Spectrosc., 25, 477 11971). ,--. -
(38E) Mitchell, D. G., Johansson, A., S ectrochim. Acta, 25B, 175 (1970). (398) N orris, J. D., West, T. S., Anal. Chim. Acta, 55,359 (1971). (40E) Omenetto, N., Met. Ztal., 61, 349 I 1 SfLl). \ - - - - I -
(41E) Shull, M., Winefordner, J. D., ANAL.CHEM.,43,799 (1971). (42E) Shull. M., Winefordner. J. D.. Avvl. Svectrosc:. 25. 97 (1971): (43Ej SyEhra, V.; Idatousek, ‘ J., Anal. Chim.Acta, 52,376 (1970). (44E) Svchra. V.. Matousek., J.., Talanta. 17,’36$ (1970). ’ (45E) Sychra, V., Slevin, P. J., Matousek, J., Bek, F., Anal. Chim. Acta, 52, 259 (1970). (46E) Thompson, K. C., Spectrosc. Lett., 3,59 (1970). (47E) Vitkun, R. A., Poluektov, N. S., Zelyukova, Yu. V., J. Anal. Chem. USSR, 25,406 (1970). (48E) Warr, P. D., Talanta, 17, 543 (1970). (49E) Zbid., 18, 234 (1971). (5OE) West, T. S., Spectrosc. Lett., 2, 179 (1969). (51E) Willis, J. B., ibid., p 191. ’
Fluorometric Analysis Charles E. White, University o f Maryland, College Park, Md. 20742
Alfred Weissler,’ Food and Drug Administration, Washington, D.C. 20204
T
is 13th of a series of biennial reviews on Fluorometric Analysis and covers the period from approximately December 1969 to December 1971 (1018). Atomic fluorescence and fluorescence X-ray emission analysis are another part of Analytical Reviews and are not included here. I n the 1970 Review, five new or revised books were listed as important additions to the literature of fluorescence analysis. Several more books have now been added. Pesce, Rosen, and Pasby (717) title their book “Fluorescence Spectroscopy” and intend it as an introduction to the technique of fluorometric analysis for biology and medicine. The theory and practice of fluorescence analysis is well covered but practically no procedures for analysis are included. Berlman has published a second volume of “Handbook of Fluorescence Spectra HIS REVIEW
Alfred Weissler is author of the Organic and Biological Section. 182 R
of Aromatic Molecules” (81). This is a welcome addition to the useful material of the first volume. A text by Becker on “Theory and Interpretation of Fluorescence and Phosphorescence” is written from a physical chemistry standpoint and provides good background material (67). In a book on “Enzymatic Methods of Analysis,” Guilbault (554) describes a number of fluorescence analytical procedures involving enzymes. A book by Winefordner, Schulman, and O’Haver titled “Luminescence Spectrometry in Xnalytr ical Chemistry” was published in February 1972. This book covers fluorescence, phosphorescence, and atomic fluorescence and is certain to be a valuable addition to the analytical literature (1058). A number of chapters in books and review articles on special phases of fluorometric analysis are of general interest. Workers in the life sciences will be interested in a chapter on Fluorometry and Phos-
ANALYTICAL CHEMISTRY, VOL. 44, NO. 5, APRIL 1972
phorimetry in clinical chemistry (790). A 45-page chapter on the theory of. luminescence is of general interest (818). A book in German on Possibilities of Instrumental Analysis contains a chapter on fluorescence and phosphorescence (79). A review article on Luminescence Spectroscopy has 143 references (664). Other articles of general interest cover fundamental concepts and applications of both spectrophotometry and spectrofluorometry (416) and fundamental concepts of fluorescence and phosphorescence spectrophotometry (694). Fundamentals and applications of fluorescence in pharmscy are treated in a seven-page article in German (946). A siummary of a lecture on Some Recent Advances in Fluorimetric Analysis by Bridges a t a joint meeting of chemical sections in England covers the general considerations with many specific examples (124). A paper on the effects of chemical structure on fluorescence which tabulates the re-
sults of various substitutions on benzene is of interest to both organic and inorganic analysts where chelation is involved (1034). The fluorescence and phosphorescence properties of 29 antioxidants and UV absorbers, which are usually added to polymers are tabulated (488). Quantum yield has been the subject of a number of articles. The compound 5-dimethylaminonaphthalene-i-sulfonic acid is advocated as an easily obtainable excellent standard for quantum yield determinations. This compound is stable, not quenched by oxygen, and gives a 0.36 value a t pH 7-9 in 0.1M NaHC03 (401). A 25page review of the measurement of quantum yields covers various aspects oi the determination of yield values (225). The quantum yield of a fluorescent compound is shown to depend upon the viscosity of the solution (841), the exciting wavelength (477, 479), the dimerization (615), and other factors. Formulas have been derived which allow the calculation of quantum yields of strongly absorbing solutions (323). Both temperature (42) and concentration (567) are shown to nave an effect on the fluorescence spectra of organic compounds. The temperature also affects the degree of polarization of the fluorescence. Corrections for the nieasurement of fluorescence polarization is the subject of a ten-page article (160). An unexpected interference in low temperature analytical fluorometry goes under the name of the Shpolskii effect which is described as a quasilinear spectrum obtained from frozen crystals under certain conditions. This effect is described in some detail (700). Several articles on different types of quenching of fluorescence deal with concentration quenching (701), oxygen quenching (422), and quenching by simultaneous irradiatlon with two short pulsed light beams (:711, 7f.2). A review with 27 references deals with the kinetics of quenching of fluorescence in small molecules (897) Lasers are finding use in fluorescence analysis, especially where conventional methods are riot easily applied. For example, a laser microprobe is used for semiquantitative analysis (1017), and infrared fluorescence spectroscopy excited by a laser beam is discussed as an analytical tool (369, 588, 595). A new book on Laser Spectroscopy is useful to those interested in laser excitation for fluorescence analysis (226). Spectrochemical fluorescence radiometric analysis (I 067) and sonoluminescence (339, 934) illustrate additional possibilities for fluorescence excitation in analytical procedures. APPARATUS
A book of 202 pages on apparatus for fluorescence analysis (461) has been
produced in the USSR. A 66-page review article on theory, instrumentation, and analytical applications of fluorometric analysis is comprehensive and has 213 references (990). Articles on diffraction gratings (555) and monochromators contain helpful information in the use of fluorometers ( 1 4 ) . A discussion of instrumental errors in measurement of fluorescence and polarization is also pertinent (697). An article on five aspects of measurement of luminescence contains useful suggestions on slit widths and other items (953). Photomultiplier tubes are thoroughly discussed and curves are given for the response of four commonly used tubes (420, 619, 880). For those who wish to build a spectrofluorometer an article describes the construction of an instrument self-correcting for both the excitation and emission spectra (671). Several of the instrument companies have added attachments to their present spectrofluorometers or produced new ones to provide automatically corrected spectra (13, 714, 977)* A number of special purpose fluorometers are as follows. The Laboratory Data Control Co. calls their fluorometer a fluoroMonitor since it is designed to measure the fluorescence of column effluent (527). This instrument is said to be sensitive to 10 picograms of quinine sulfate. The cell volume is 3 pl. The Farrand Optical Co. Inc. advertises a microscope spectrum analyzer for “quantitative microscopic fluorescence and absorption studies’’ (278). Fisk Associates (293) have designed an automatic calcium titrator for the fluorometric determination of Ca where the fluorescent Ca-Calcein complex is titrated with EGTA (ethyleneglycoltetracetic acid). The American Instrument Company has introduced a Chem-Glo photometer for rapid ATP determinations. The instrument is designed for bioluminescent and other chemiluminescent reactions (12). For laser-induced fluorescent studies, a continuous wave chemical laser has been. designed (899), and Texas Instruments has provided a cathodeluminescence attachment for microprobe analysis (766). 4-n inexpensive therrnostated cell compartment has been described as a n accessory for the Aminco-Bowman Spectrophotofluorometer (937). A simple cuvet holder and rinser are described by these same authors (936). They also have devised attachments for filter paper supports and capillary droplet arrangements (938). An accessory for the Unicam SP860 spectrofluorometer allows a read-out of the excitation spectrum produced by the fluorescent substance (238). A Dewar sample cell assembly for low-temperature fluorometry is a useful accessory (486).
Flexible fiber optics have been applied with a rotating sample holder at liquid helium temperatures (978). ,4 number of papers have been concerned with improving apparatus for the measurement of weak luminescence ( f 9 7 , 254, 478). Sensitive apparatus for use in chemiluminescence analysis has been improved (625, 800). Apparatus for fluorimetry of a thin liquid layer is described along with the theory and performance of the system (936). Digital recording has been successfully applied to fluorescence recording systems (551). Specifications and diagrams are given for a highsensitivity flowcell system for the autoanalyzer (11). The design and performance for a differential spectroApfluorometer is reported (46). paratus combining a gas chromatograph with a spectrofluorometer has been successfully applied (86, 138). A new scanner has been described for fluorometric quantitation of thin layer chromatograms (43.2). A device for measurement of the intensity of a small part of the spectrum in fluorescence analysis by selection with interference filters is described (1.60). An unusual situation is described in a patent for a n optical system of determining fluorescence emitted by excitation with sunlight (580). Another simple device is one for end-point detection in fluorometric eiid-point titrations (178). Fluorescence polarization apparatus is the subject of several articles concerning equipment. A homemade polarization fluorometer has been designed and tested (781). A spectrofluorometer-polarization fluorometer combination is reported to record corrected excitation and continuouspolarization spectra and also transmission, as a function of wavenumber (10.41). A device for rotating film holders is described for holding the film for polarized fiuorescence measurements (7’92). An application of polarization that may prove of value is the use of polarizers in separating the exciting radiation from the emission. This method is applicable only to unpolarized emission (1055). A USSR patent has been granted for a fluorometer equipped with spectrum converters and designed for high speed in biological applications (245). An article on the design and principles of fluorescent radiat.ion converters is informative reading on this topic (472). A fluorometric attachment for an at,omic absorption spectrometer is designed for the determination of uranium by the N a F fusion method (129). Glass standards for the U determination by NaF fusion are now available (38). A demountable excitation unit for cathode luminescence spectroscopy is reported in an article giving also the yttrium spectrum from yttrium garnet (16).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 5, APRIL 1972
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An instrument has been designed for studying electroluminescence and also sonoluminescence of liquids (338). INORGANIC
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General. Since fluorescence analysis is capable of measuring parts per billion of some elements, it is appropriate t o start this section with a reference t o an article on the preparation of ultrapure water (418). A general paper on solution luminescence of metal complexes with 62 references is also in order (666). A study of the polyhydroxy flavones as extraction reagents shows that many of the metal complexes with morin can be extracted with isoamyl alcohol. Curves and tables are given to show the optimum pH for the extraction of the morin chelates with about 40 elements. The color of the fluorescence of the organic solution is also given (97). Quercetin with 2% fluorescent green as an indiacator is recommended as a spray reagent for metal ions in thin layer chromatography on silica gel; colors and limit of detection are given for 17 elements (440). Since 8-quinolinol is used extensively in chelating for inorganic analysis, an article showing the effect of temperature, from 77 O K to room temperature, and concentration on 5- and 8-quinolinol is of interest (336). The fluorescence of 8-quinolinol in strongly basic solutions has also been studied (819). The use of fluorescence in determining formation constants of 8-quinolinol metal complexes is under study ( 9 3 ) . The limit of detection of 20 cations on filter paper has been determined on spraying with 8-quinolinol and with tetramercuriated fluorescein. I n the latter case, the paper is first treated with H2S and followed by a spray with the reagent. Under UV irradiation, the metal ion locations appear as dark spots with the mercuritluorescein, and with 8-quinolinol the cations appear as light spots (1047). Possible structures are proposed for the bivalent metal chelates of o,o'dihydroxyazobenzene (498) The luminescence of 2,2',2"-terpyridine and some of its chelates has been the subject of a doctoral thesis (289). The scanning system of an electron microscope has been used for the cathodiluminescence study of a number of minerals and the wavelength of maximum emission intensity is given for six minerals (200). A fine research on the tetracyanoplatinates(I1) compounds has shown that the tetracyanoplatinates (11) of Y, Zr, Ag, Zn, Cd, Hg, All Pb, and T b are fluorescent and the limit of detection by this means ranges from 5 to 200 ppm. Hf yields a nonfluorescent salt but the Zr salt is fluorescent; hence,
184R
20 ppm of Zr can be detected in Hf (14.6). Aluminum, Beryllium, and Boron. Salicylaldehyde can be used to fluorometrically determine A1 in the presence of Be in about 1O-sM concentrations of each by using a wavelength slightly below the excitation maximum; or the two elements may be separated by paper chromatography with 2M HC1 saturated BuOH prior to spraying with 5% of the salicylaldehyde in alcohol (76). Research on the use of hydrazones in fluorometry has shown that 2 hydroxy-1-naphthaldehyde benzoyl hydrazone may be used as a reagent for A1 from 0.1 to 1.0 pg/ml using an excitation at 395 nm and emission at 475 nm (986). Lumogallion is reported to be a superior reagent for the determination of A1 in sea water because considerable fluoride and phosphate may be tolerated (863). I n the analysis of low pressure polyethylene after ashing with 50% HzOz, the A1 was determined fluorometrically with 8quinolinol and the Ca with fluorescein complexion (612). The A1 in sodium hydroxide from a n Hg electrolysis was also determined by the 8quinolinol fluorometric method (999). Morin solution sprayed on filter paper spots from the ring oven technique has been used to analyze air pollution samples for A1 (61). Morin has also been used for fluorescent localization of A1 in plant tissues (261). Two new reagents have been suggested for Be. Tetracycline along with 5,5-diethyl-2-thiobarbituric acid gives a blue-violet fluorescence with Be at p H 9 in a borate bufEer. A linear response is produced with 5 to 15 pg of Be per 50 ml. In the complex, Be and the two organic compounds have a 1 : 1:l ratio. The ions Fe(III), All Ca, and Ba in 2-fold amounts relative to Be interfere. The fluorescence is measured a t 506 nm with an excitation at 406 nm (641). The compounds salicylidene-2 -amino - 3,5 - dimethylphenylarsonic acid and salicylidene-2-aminophenylarsonic acid have been studied in reactions with 28 metal ions. The first of these two compounds is described as a reagent for Be ( 4 1 1 ) . I n a study of the metal chelate of flavone derivatives, it was found that the Be complexes with 3-hydroxy-4-methoxy-, and 3,5,7trihydroxyflavone gave fluorescent chelates with Be with greater intensity emissions than the others used (387). Another new reagent for Be, 2-ethyl-3methyl-5-hydroxychromon1 forms a fluorescent complex with Be which has its maximum excitation a t 403 nm and emission a t 483 and is extracted by CC14 a t pH 6.5-10; phosphate, fluoride, and several metal ions interfere (@6). Another extraction method for Be in natural waters in the order of 1 pg of BeO/ml uses the acetylacetonate-trylon B complex by CHCll extraction (621).
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Three interesting reports have evolved from a continued research on the analysis for boron with a combination of polyhydroxy flavones, such as morin with oxalic acid. The sample in MezCO is treated with an ether solution of oxalic acid and also an ether solution of morin. The ether is removed in vacuo and the residue is heated to 110 "C for 20 min. The residue is dissolved in Me&O and diluted with ether. After 30 min., the solution was excited a t 430 nm and its intensity read a t 518 nm. The linear result is from O.OWO.02 pg/ml (209, 760). A possible structure of the boron complex is proposed (761). Quercetin and kaempferol react with oxalic acid and boric acid in a manner similar to morin (960). Current research is also concerned with the effect of substituent groups on the fluorescence of the boron polyhydroxyflavone complex (662). Boric acid also forms a fluorescent complex with dibenzophenone in concentrated sulfuric acid with a calibration graph 0.05 to 0.6 p M of H3B03 (679). Another reagent for boron, 2-hydroxy4-methoxy4'-chlorbenzene, is said to give good results a t 74 ppb to 5 ppm; this reagent has been used to measure directly the boron content of natural waters (647). Calcium, Barium, Lithium, Magnesium. The ions of Ca, Sr, and Mg are extracted with a mixture of phenyltrifluoroacetone and Rhodamine S by benzene. The complexes have an absorption maximum of 540 for Ca and Sr and 544 for Mg and an emission maximum of Ca and Sr at 565 and Mg a t 570 (734). Tetracycline serves as a fluorescent indicator for fluorescent end point on titration of Ca, Sr, Mg, Cd, or Zn with EDTA. From 0.4 to 1.6 mg can be titrated with 0.4mM to 0.176M EDTA. The method is less sensitive for Cd and Zn. A procedure for Ba is also given (34). A comparison of eight different indicators for the fluorometric end-point chelatometric titration of Ca in the presence of Mg has been reported along with a description of optimum conditions (233). Statistics on the fluorometric determination of Ca with different workers have been compiled (186). Automated fluorometry for Ca can determine 0.2 pg/ml in a 20 pl sample (667). Serum calcium titrated with calcein is also automated (688). Lithium has been found to form a 1 : 1 complex with 5,7-dibromo-8-quinolinol that gives a stronger fluorescence than the 8-quinolinol complex. The absorption and emission curves are given and the apparent formation constant is 3.6 X 105. The optimum conditions are an ethanol solution pH 9-9.5 and a Li content of 1-100 pg/4 ml; Fe(II1) interferes but Ca, Sr, Ba, Al, Mg do not (738). No new reagent has appeared for Mg but one paper shows the effect
of Ca and phosphate on the automated determination of Mg in biological fluids (864)
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Copper, Silver, Gold. Thiamine as a reagent for copper after reduction t o Cu(1) has been further studied and interferences have been determined; the sensitivity is given as 0.1 pg/ml (1052). The determination of 1 ng of Cu in 2 ml of solution can be accomplished with the etioporphyrin complex a t -196 "C in heptane, however Zn gives a similar reaction. Interferences are listed (877). The catalytic reaction of traces of Cu with luminol and HzOz is sensitive up to 0.05 ppm (249). Another chemiluminescent method for copper uses N-(6-hydroxylpropy1)-anabasine in HZOZpH 10-11 solution The cupric ion causes a decrease in intensity and a shift of the emission to shorter wavelengths. The analytical range for this reagent is 0.5 to 0.001 pg/ml (1068). The discovery of cysteine as a reagent for Cu occurred in an interesting fashion. ' 4 fluorescence in skin extracts was traced to copper from the homogenizer and cystine on the skin sample. Au and Ag ions also form fluorescent complexes with mercaptides. The emission range is 530-60 nm and the excitation 250375 nm. The emission maximum depends on both the ion and the ligand (19). A quenchometric titration of cupric ions with EDTA with 3-carboxy-7-hydroxy coumarin as a n indicator will determine 0.06-6 pg of copper (482). Aromatic carboxylic acids serve as fluorometric reagents for both copper and vanadium (501) . The fluorescent complex formed with silver ions in a mixture of 1, 10-phenanthroline and eosine is extracted with a mixed solvent of acetone and ether for the determination of silver in minerals. An analytical curve is given for 0.5-10 pg of Ag in 6 ml of extract (553). The nitrogenous bases from some petroleum oils have been shown to be good fluorescent adsorption indicators for the determination of silver (925). Research on kojic acid as a reagent for gold has continued; the linear fluorescence intensity curve is reported as 0.5 to 20 pg of Au and the sensitivity is listed as 1 pg; Fe and Te interfere (729). The Rhodamine dyes have been studied extensively as reagents for h u (732), and a favored one is para-dimethylaminobenzylidenerhodamine. The absorption maximum is a t 44 nm and the emission 560 and 580 nm, and the analytiral curve is linear to
