ANALYTICAL CHEMISTRY, VOL. 50, NO. 5, APRIL 1978
189R
Fluorometric and Phosphorometric Analysis C. Michael O'Donnell" Clinical Chemistry Laboratory and Department of Pathology, University of California,
Los Angeles, Los Angeles, California 90024
T. N. Solie" Departments of Physiology and Biophysics, and Chemistry, Colorado State University, Fort Collins, Colorado 80523
This is t h e sixteenth of a series of biennial reviews on fluorometric and phosphorometric analysis. T h e literature cited was indexed by Chemical Abstracts during the period from 1 December 1975 to 21 November 1977 and has appeared since the last review ( 6 7 A ) . Owing to the very large number of publications in the area of luminescence spectroscopy, selection of citations to be included in this review was necessary. T h e selection process evolved in three stages: (1) key word index of Chemical Abstracts, (2) reading of the title and abstract. (3) inclusion in the journals listed in Table I, and (4) content of the article. An attempt has been made to select articles of analytical interest. Books and reviews were selected for a broad coverage in many related areas of luminescence spectroscopy. Applications in biophysical chemistry and polymer chemistry have been cited only when the papers focus on instrumentation or data analysis. Applications involving many aspects of electronic molecular structure have been excluded as well as the area of solid-state inorganic luminescence. Publications in foreign journals not readily available have generally not been cited. Atomic fluorescence, bioluminescence, chemiluminescence, and x-ray fluorescence appear in other Analytical Reviews and are not included here. Undoubtedly some of the selections reflect the authors' interests. Any omission of work advancing t h e applications of fluorometry a n d phosphorometry is regretable but perhaps inevitable in the effort to keep the number of citations to a number acceptable t o ANALYTICAL CHEMISTRY. T h e bibliography is divided into fourteen sections with separate numbering for each. Books and Reviews (A), Instrumentation and Data Processing (B), Kinetic Spectroscopy: Quantum Yields, a n d Lifetimes (C), and Luminescence Combined with Separation Methods (D) appear first because of their general interest. The remaining sections are composed of selected applications: Amines (E); Amino Acids, Peptides, and Proteins (F); Clinical Chemistry (G); Environmental Chemistry (H); Enzymes (I); Food Science (J);Inorganic Chemistry (K); Miscellaneous Organic Compounds (L); Pharmacology (M); Purine, Pyrimidines, and Nucleic Acids (N).Each citation includes the title in English. These titles are intended to aid the reader in attaining maximum benefit from this compilation. Comments in the text indicate the direction of development in particular aspects of fluorescence and phosphorescence. A comprehensive critical survey of developments has not been attempted.
GENERAL Instrumentation. T h e increasing availability of low to moderate cost commercial instrumentation for luminescence spectroscopy has made fluorometric and phosphorometric methods of analysis accessible to virtually all laboratories desiring to use them. Other instrumental methods of comparable cost rarely meet the analytical sensitivity of fluorometry and phosphorometry. It seems clear that, a t least, fluorescence spectroscopy is in the rapidly growing stages three through five of the proposed seven stages in the development of analytical methods (H. A. Laitinen, Anal. Chem., 45, 2305 (1973)). In addition, there seems to be a n unparalleled cooperation and communication among the measurement specialists and the originators of the analytical problems; a desirable situation for complex problems (H. A. Laitinen, Anal. Chem., 49, 1889 (1977)). 0003-2700/78/0350-189R$05 O O / O
General aspects of fluorescence and phosphorescence spectroscopy have been summarized by recent books and reviews. T o note a few, we mention the book by Schulman (77A)and the collections edited by Chen and Edelhoch (17A, 18A), by Birks (IOA) and by Wehry ( 9 I A ) . Many other excellent reviews are listed in the bibliography under Books and Reviews. T h e reader is invited to browse through the titles for citations of particular interest. Recent advances in instrumentation can be viewed as developments in (a) excitation sources with or without dispersive systems, (b) detectors, and (c) data handling methods and procedures. Reviews of these topics include those by Naqui (101B) and Soutif (1323). Double-beam fluorescence spectrometers (2B,113B) have become commercially available. These instruments frequently improve quantitative measurements by reducing interferences from scattering and unwanted fluorescence. Instrumentation for differential fluorescence measurements has been described by Bostian (13B) and by Bablouzian and Fasman ( 4 B ) . Unprecedented gains in sensitivity are being realized using photon-counting spectrometer systems for continuous fluorescence (77B, 79B) and for fluorescence decay (9B, 38A). Other developments in specialized instrumentation have included slit-lamp fluorometers (5OB),fiber-optic fluorometers (IOOB),optical summing spectrometers (70B), and multichannel microspectrofluorometers ( 8 I B , 8%). Light Sources. A comparison of pulsed source excitation with continuous wave source excitation in molecular luminescence spectrometry has been presented by Winefordner's group (15B).They report that in general, pulsed-source excitation does not offer significant signal to noise advantages over continuous wave excitation and detection unless the major source of noise can be associated with the detector system or with nonsource induced background. The noise originating from the source, the sample and/or the detection system limits the sensitivity of any spectrometric system. Talmi et al. (139B) have considered the limitations of several common afmlytical spectrometric sources. There is considerable activity in adopting lasers as excitation sources (4OA, 16B, 59B, 143B); . Sample Optics. A thirty-fold increase in sensitivity for a fluorescence spectrometer is reported to be obtained by rotating the monochromator slit images by 90' to permit viewing the sample along the length of the excitation slit image; then adding concave retromirrors behind the sample in both the excitation and emission beams (15OB). Other efforts to improve sensitivity and/or ease of operation a t the sample compartment have been considered; geometry of the cuvette (105B),flow cells (130B),size of the sample (121B, 122B) and Dewar vessels (I02B). Detectors. In a comparison of imaging devices with photomultiplier tube detectors, Winefordner's group (25B28B) found that the silicon vidicon was not analytically useful, in terms of signal to noise, for molecular luminescence spectrometry but that the integrating image devices do offer analytical potential in combining multichannel time advantage with signal to noise approaching that of photomultiplier tubes. Developments in video-spectrometer systems include rapid scanning fluorescence spectrometers (80B) and derivative spectrometers (24B). Simultaneous split beam ratio measurements (34B, 78B, 147B) and other electronic modifications (45B, 54B, 60B, 62B) are directed a t improving the signal to noise ratio for detecting weak luminescence signals. C 1978 American Chemical Society
190R
ANALYTICAL CHEMISTRY, VOL. 50, NO. 5, APRIL 1978
Table I. Journals Chosen for Compilation of Citations in Chemical Abstracts, Volumes 84-87 Acta Chem. Scand. Analyst ( L o n d o n ) Anal. Biochem. Anal. Chem. Anal. Chim. Acta Anal. Lett. Angew. Chem., Int. Ed. Appl. Opt. Appl. Spectrosc. Atmos. Environ. Aust. J. Chem. Biochem. J. Biochem. Med. Biochim. Biophys. Acta Br. Med. J. Bull. Chem. SOC.Jpn Bull. Environ. Contam. Toxicol. Bull. Narcot. Bunseki Kagaku ( J p n Anal.) Can. J. Biochem. Can. J. Chem. Can. J. Pharm. Sci.
Can. J. Spectrosc. Chem. Br. Chem. Ind. Chromatographia Chromatogr. Rev. Clin. Biochem. Clin. Chem. ( Winston-Salem, N.C.) Clin. Ch im. Acta Environ. Lett. Environ. Sci. Technol. Forens. Sc i. Fresenius’ Z. Anal. Chem. Helv. Chim. Acta Indian J. Appl. Chem. Indian J. Chem. Indian J. Pharm. Indian J. Technol. J. Agric. Food Chem. J. Air Pollut. Control Assoc. J. A m . Chem. SOC. J. A m . Oil Chem. SOC. J. Assoc. Off. Anal. Chem.
Data Processing. Automated luminescence spectrometer systems using an on-line minicomputer (155B) or a microprocessor (74B) for correcting and recording absorption, excitation, and emission spectra are greatly extending the qualitative and quantitative analytical capabilities of the method. Warner et al. (144B, 145B) have developed a method involving a data matrix derived from multiple-wavelength excitation-emission spectra for the analysis of fluorescent samples containing multiple components. These authors suggest that five components with strongly overlapping spectra can be determined simultaneously with an accuracy of 5% or better. Wavelength modulation techniques (IIOB)and other computer assisted methods (117B) have been used in fluorometric analyses of mixtures without separation of the components. Computer assisted methods for qualitative analysis and chemical characterization based on fluorescence spectra have been reported by Miller and Faulkner (98B, 154B). A computer based file searching procedure was developed to compare chemical structural features with characteristics of the fluorescence excitation and emission spectra. Substantial spectral data compression could be achieved by a Fourier transformation of the fluorescence spectra, followed by use of limited arrays of the Fourier components in the search. This approach offers substantial promise if large collections of standardized corrected spectra can be obtained. S t a n d a r d s a n d Calibration. A calibrated tungsten lamp in a silica envelope for obtaining spectral sensitivity curves for the calibration of spectrometers is described by Parker (109B). Reference materials and methods for instrument calibration have also been discussed by others (35B,52B, 95B, 97B). Lifetime Spectroscopy. Lifetime measurements of electronic excited states yield dynamic information about the photoexcited state (60C). I t is now possible, using modern instrumentation, to obtain lifetimes with resolution comparable to high resolution absorptivity measurements. Instrumental methods for measuring lifetimes include phaseshift techniques (73B, 124B), chopper techniques (35C) and time-correlated single-photon counting techniques (38A,64B, 5C, 23C, 31C); the latter being perhaps the most widely used. Potential limitations of the various methods for lifetime measurements have been discussed by several workers ( 126B, 127B, 6C, 30C). In the analysis of experimental fluorescence time decay data, the moment index displacement procedure developed by Isenberg (128B, 129B) provides for an automatic correction of three nonrandom instrumental errors (41B),if the decays can be treated as simple exponentials or sums of simple exponentials. The theoretical origin of non-exponential fluorescence decay has been discussed by Langhoff (44C). Luminescence Combined with Separation. There have been many attempts since the last biennial review to combine fluorometric and phosphorometric techniques with various separation methods. The range of applications is suggested by the titles in the bibliography section D. Some specific pairings are with high-performance liquid chromatography
-
J. Chromatogr. J. Chromatogr. Sci. J. Food Sei. J. Forens. Sei. Soc. J. Indian Chem. Soc. J. Pharm. Pharmacol. J. Pharm. Sei. J. Sei. Food Agric. J. Steroid Biochem. Microchem. J. Mikrochim. Acta Nature, London Naturwissenschaften Pestic. Sei. Photochem. Photobiol. Rev. Sei. lnstrum. Scand. J. Clin. Lab. Invest, Science Sep Sei. Spectrosc. Lett. Steroids Talanta
( 2 0 , 11D, 25D), thin-layer chromatography ( 4 8 0 ) and polyacrylamide electrophoresis (16D). There are many others. Indeed it is difficult to estimate the extent of the activity because many of the applications are simply described as experimental methods in the particular work and are not indexed under the key words chosen for the search of chemical abstracts.
APPLICATIONS Selected comments are used in this section to indicate progress in utilizing some of the newer analytical techniques of luminescence spectroscopy. Although the majority of applications reviewed demonstrate the continuing success of simplicity in luminescence analysis, the power of combining luminescence and separation technology is readily apparent in the many accounts of this combination appearing in the literature. The resolving power of combining a technique such as high performance liquid chromatography with a multichannel fluorescence spectrometer approaches that available in the most sophisticated analytical equipment presently available. An area in which the authors anticipate a significant increase in activity over the next few years is the area of fluorescence labeling in immunoassays. These assays are the fluorescence counterparts of the radioimmunoassay techniques which are based on a competition between antigen and labeled antigen for antibody combining sites. The equilibrium equation describing this competition may be written Ag
+ Ag* + Ab + AgAb + Ag*Ab
where Ag* = labeled antigen, Ag = antigen, and Ab = antibody to ‘4g. Fluorescent labeled antigens have several advantages over radiolabeled antigens because they are relatively stable and are not restricted by regulations such as those governing the transport, handling, and storage of radioactive materials. The disadvantages of fluorescent labels are their susceptibility to quenching, photolability, and lack of commercially available instruments suitable to handle the varied approaches to fluorescence immunoassays. Fluorescence polarization has been used in the immunoassay of the aminoglycoside gentamicin (93G) based on the polarization changes when the fluorescein labeled gentamicin binds t o the large molecular weight antibody. An alternative approach to the immunoassay of gentamicin utilized umbelliferyl-@-galactosidegentamicin which served as a substrate for bacterial &galactosidase ( I l C ) . The labeled gentamicin substrate was inactive when bound to an antibody but is subsequently reactivated when displaced by unlabeled gentamicin. Hence, the rate of production of fluorescence was proportional to the gentamicin concentration. The principles of fluorescence excitation energy transfer have been applied to the analysis of drugs (89G). A fluoresceinlabeled antigen and a quencher (rhodamine) labeled antibody
ANALYTICAL CHEMISTRY, VOL. 50. NO. 5, APRIL 1978
C. M. O'Donnell is the Assistant Director of the Clinical Chemistry Laboratories and an Assistant Professor in the Department of Pathology at the University of California, Los Angeles, Center for the Health Sciences. Born and raised in Louisiana, he studied chemistry at Louisiana State University, New Orleans, and received a B.S. in 1962. He obtained a Ph D. in physical chemistry from Louisiana State University, Baton Rouge, in 1966. After a year of postdoctoral study at the University of New Mexico, he joined the facuky at Colorado State University in 1967. He was an NIH postdoctoral fellow in clinical chemistry at the University of Colorado Medical Center in Denver. He then joined the staff at Bic-Science Laboratories in 1974 before accepting his present position in December 1975. His research interests have been in the areas of fluorescence and phosphorescence spectroscopy including photochemistry. Recently his interests have been in biochemical applications particularly the application of fluorescence techniques in the discipline of clinical chemistry. He is a member of the American Association of Clinical Chemists, The International Association of Forensic Toxicologist, California Association of Toxicologists, Phi Lambda Upsilon, and Sigma Xi.
1. N. Solie is Associate Professor in the Departments of physiology and Biophysics, and Chemistry at Colorado State University, Fort Collins, Colo. Born and reared in Minnesota, he studied chemistry and mathematics at the University of Minnesota, Minneapolis, and received a E.A. in 1959. He obtained a Ph.D. in physical chemistry from the University of Oregon, Eugene. He was a postdoctoral fellow in biophysics at the University of Colorado Medical Center, Denver, and in molecular biology at Vanderbilt University. Following a year as assistant professor of physical chemistry at Luther College, Decorah, Iowa, he joined the faculty at Colorado State University in 1967. His research interests are an assortment of biophysical problems, including applications of time resolved phosphorimetry and fluorimetry. He is a member of the Biophysical Society, AAAS, American Chemical Society, and Sigma Xi.
were employed. In the absence of unlabeled antigen the labeled-antigen is quenched. Quenching can be inhibited by the addition of unlabeled antigen in proportion to the concentration of unlabeled antigen. Fluorescence immunoassays have also been reported for pesticides (22H),the C3 component of human complement (12G),and serum IgE ( 1 i G ) labels. A new analytical technique was developed for the analysis of gas phase concentrations using photon catalysis (12K). This method requires injection of an excess of an energetic metastable species into a gas stream containing the analyte. Energy transfer takes place from the metastable species to the analyte, resulting in excitation and subsequent fluorescence characteristic of the analyte with an intensity proportional to analyte concentration. A silicon-intensified target (SIT) image detector combined with an optical multichannel analyzer (OMA) was used as the detector for the analysis of phosphorus monoxide in flames ( 2 9 K ) . An OMA detector was coupled to a fluorescence spectrometer to monitor effluent peaks in the liquid chromatographic analysis of petroleum fractions (1IH).T h e results of these studies indicate that although multichannel detection of fluorescence is in its early stages of development, it may find many applications with improvements in the development of data handling routines. Synchronous excitation fluorescence spectrometry is helpful in the analysis of environmental contaminants. Ordinarily a fluorescence spectrum is recorded a t a constant excitation wavelength. In synchronous excitation fluorescence spectrometry, the excitation monochromator is allowed to vary while maintaining a constant wavelength interval between the excitation and emission wabelengths. An enhanced selectivity arises from the differences in the excitation spectra of the components in a mixture. This technique has been applied to the analysis of crude oils (14H, 1 5 " ) . tracer measurements and in drug analysis (2M). in hydrology (lH),
191 R
Laser excitation sources are being utilized to increase both the sensitivity and selectivity of fluorescence analysis. Laser-induced fluorescence has been applied to the sub-partper-trillion detection of polycyclic aromatic hydrocarbons (34L). T h e limits of detection for pyrene was 0.5 ppt,. Attaining reproducible results a t trace levels such as this requires careful attention to sampling. reagents, and sample positioning. An interesting approach to combining laser excitation with HPLC has been reported for the analysis of aflatoxins B1, B2, G1, and G2 ( 9 4 . The laser source was focused onto a small spot inside a droplet emerging from the chromatographic column. T h e liquid droplet formed a windowless fluorescence cell, which avoided the problem of fluorescence from cell walls. Although standards could be analyzed down to 750 fg for the four aflatoxins, extracts of corn were found to possess interfering substances which limited detection to approximately 2 ppb. A new technique has been developed for trace analysis by selective excitation of probe ion luminescence (SEPIL) (27K, 45K, 77K). This method is capable of detecting lanthanide ion concentrations a t 25 parts in 1015 with appropriate incorporation into a CaF, lattice. A similar approach may be utilized for nonfluorescent ions such as Pod3-ions which perturb the localized crystal fields of Eu3+ ions enough to permit selective excitation of the perturbed sites in the presence of nonperturbed Eu3+ ions (77K). Although the limits of detection for PO,$3-were not particularly good (0.19 g/mL), this approach may yet prove useful with further optimization of the lattice and for application to other ions. T h e use of lasers will undoubtedly provide increased sensitivity (19J,20L) and selectivity over conventional excitation sources but the demands for reagent purity, prevention of sample contamination, and minimization of scatter and other experimental interferences will correspondingly increase. Although many molecules luminesce, a large percentage of organic molecules either do not fluoresce or have fluorescent quantum yields so low as to make fluorescence analysis inapplicable. For this second group of molecules. fluorescent tags often provide a solution and much work has been done particularly in fluorescence studies of proteins. Labeling of gamma globulins with fluorescamine and 2-methoxy-2:4-diphenyl-3(2H)-furanone (MDPF) was investigated (24F). T h e authors developed a method to measure the ratio of fluorogen to protein and determined the effect of this ratio on antibody specificity. .4n automated procedure for the assay of proteins and peptides was described utilizing 0-phthaldialdehyde as a label ( i 6 f l . As little as 2.4 pmol of bovine serum albumin may be detected with this label and appears to offer some advantages over fluorescamine. Anthroyl stearate was the first non-amine fluorescence probe of chloroplast membranes used (80F). This probe may be used to study proton gradients and inhibition of these gradients by reagents in chloroplast. T h e principles of competitive binding were applied to the assay of ligands by monitoring the enzymatic hydrolysis of ligand-fluorescent dye conjugates 19F). This assay requires the use of nonfluorescent conjugates which act as substrates in an enzyme-catalyzed reaction which produces fluorescent products. The enzymatic reaction was inhibited by a binding protein or specific antibody but proceeded when the substrate was displaced by the addition of unlabeled ligand. This method measured ligand concentrations as low as approximately 10 n M and should prove applicable to many biologically important ligands. Fluorescent labels have found riumerous analytical applications such as the assays of vasopressin and oxytocin (23F), amino acids in natural water (17H) and sugar beets ( 5 J , carcinogenic aromatic amines ( 3 2 H ) . the drug bleomycin (86G).and organophosphate pesticides (21H). New materials for labeling have been introduced, that is, 5-isothiocyanato-1,8-naphthalenedicarboxy-4-methylphenylimide for amino groups (38fl and 4-bromomethyl-'7-methoxy-coumarin for acidic herbicides ( 5 H ) . Inorganic ions can be utilized to increase the selectivity and enhance the sensitivity of fluorescence and hos horescence procedures. Divalent ions such as Co2+.Cu2f M&, and Zn2+ complex with fluorescent etheno analogues of the adenine nucleotides (1I N ) . Complexation results in the quenching of the fluorescence of the adenine nucleotides analogues and
192R
ANALYTICAL CHEMISTRY, VOL. 50, NO
5, APRIL 1978
forms the basis for the determination of enzyme activity of enzymes which are ADP or A T P dependent. Although excitation and fluorescence spectra are similar for ATP, ADP, and AMP, the fluorescence intensities of each species vary considerably. Therefore the resultant change of fluorescence intensity upon the conversion of ATP -* ADP, etc. forms the basis for the determination of enzyme activity. Cadmium(II), copper(II), mercury(II), and zinc(I1) ions were used as inorganic probes in a study of the nucleosides adenosine, cytidine, guanosine, thymidine, and uridine (2N). Enhancement and quenching effects were found to be dependent on both the individual nucleoside and the particular metal ion as opposed to the nonspecific quenching of most fluorophors by the external heavy atom effect produced by KaI. T h e specificity of inorganic probes provides another means of improving selectivity in luminescence analysis. Improvements in the kinetic-based fluorometric determination of Ag(1) has been described (76K). This method has a linear dynamic range of 6 ppb-30 ppm with good precision. Energy transfer from lead(I1) to manganese(I1) has been utilized in the analysis of traces of Pb(I1) in NaCl pellets (17M. Several matrices with varying donor and acceptor ions s e r e listed from other references to indicate the applicability of this technique. T h e NaCl matrix was found to permit the detection of Pb(I1) down to 20 ng/mL with reproducibility approximately f 5 % . It was suggested that this approach may be adapted to multielement analysis in a single pellet. T h e heavy atom effect was found useful in quantitative phosphorescence analysis at room temperature (BL,22L,45M). A filter paper impregnated with lead or thallium salts before application of the specimen (22L) permitted detection of 50 pg of cinoxacin by room temperature phosphorescence. This technique is applicable to a number of organic molecules. Sodium iodide provided enhanced sensitivity in the detection of drugs and biologically important molecules (45M). The heavy atom effect from the addition of silver nitrate or sodium iodide was found very effective in inducing phosphorescence emission (8L). Undoubtedly these effects will find increasing applicability in future work on room temperature phosphorescence analysis. Photochemical transformation of a nonfluorescent molecule into one which is fluorescent or vice versa forms the basis for an elegant analytical technique. A number of phenothiazines may be determined in the ppb range of concentrations by in-situ photooxidation and simultaneous measurement of fluorescence intensity (47M). Similar approaches have been reported for vitamin K1(14 and 2-[4-(4-chlorophenoxy)phenoxylpropionic acid (2OM). The photochemical dimerization of 12(9-anthroyloxy)-stearic acid (12-AS) proceeds by a diffusion-limited second order mechanism in many solvents (50fl. T h e resultant dimer is nonfluorescent and the decay of monomer fluorescence was used to study diffusion processes in these solvents and fluid lipids. Chemiluminescence detection continues to be applied to trace inorganic and organic determinations. The advantages of this approach in the analysis of nitrogen in petroleum fractions over microcoulometry have been observed (21K). The presence of other reducing substances interferes with the determination of glucose in urine by chemiluminescence detection. Elimination of these interferences was achieved by using a Somogyi precipitate (311) or substituting the bis(2,4,6-trichlorophenyl)oxalate-CLsystem for the luminol-CL system a t an acidic p H (301). A chemiluminescence method for the determination of lactate dehydrogenase activity has been automated utilizing the bis(2,4,6-trichloropheny1)oxalate-CL system (33L). Some additional applications of the chemiluminescent technique were the determination of trace chromium(lI1) levels in water samples (31K) and the continuous monitoring of reactive hydrocarbon air pollutants ( 7 H ) . Since these selected applications are intended to be but a small sampling of the enormous developments in fluorescence and phosphorescence analysis over the past two years, we encourage the reader to review the titles under the applications section of this paper in order to fully appreciate the direction of these efforts.
ACKNOWLEDGMENT W e acknowledge the assistance of Alice Rung6 in coinpillrig the references for this article. We appreciate the cooperation
of Beverly Lau in coordinating the arduous task of typing the bibliography and manuscript. The substantial support of our employers toward the preparation of this review is gratefully acknowledged. This work was supported. in part, by the National Institutes of Health under grant ESAM 00987-02. BIBLIOGRAPHY
A. Books and Reviews (1A) Phosphorimetry. Spectrochemical analysis. Aaron, J. J , Winefordner, J. 0.; Talanta, 1975, 22(9), 707-15 (2A) Fluorescence and its analytical application in pharmacy. Pari I. Theoretical and experimental aspects. Airaudo, C. B.; Gayte-Sorbier, A , ; Labo-Pharma-Probl. Tech.. 1975, 23(248), 1027-35 (Fr,). (3A) Fluorescence and its analytical applications in pharmacy. 3. Applications and bibliography. Airaudo. C.B.; Gayte-Sorbier, A,; Labo-Pharma-Probl. Tech., 1976, 24(251), 125--39 (Fr.) (4A) Fluorescence and its recent analytical applications in pharmacy. Airaudo. C. B.: Gayte-Sorbier. A.; &bo-Pharma-Probl. Tech., 1976, 24(254), 473-85 (Fr 1. (5A) Phosphorimetry. 1. Baeyens, W.; Pharm. Weekbi., 1976, 111(41). 1006-1019 (Dutch) (6A) Phosphorimetry 11 Baeyens W , Pharm Weekbl , 1976, 111(43) 1075-1082 Dutch) (7A) Qualitative and quantitative luminescence-bituminologicalanalysis. Baranova. T. E.:Lyumln. Bitumi,noiogiya. 1975, 34-55, 186-91 (Russ). Edited by Florovskaya, V. N.: Mosk. Univ : Moscow, USSR. (8A) Emission Spectroscopy, Barnes, Ramon M., Ed.: Publ. Halstead Press: John Wiley & Sons, Chichester. Sussex, 1976. 548 pp. (9A) The Analysis of Organic Materials. No. 1 1 : Colorimetric and fluorimetric Analysis of Steroids. Bartos. J.. Pesez. M.: Academic: London, Engl.. 1977, 274 pp. (10A) Excited States of BiologicalMolecules. Birks. John E . , Ed.; John Wiley, London, 1976, 652 pp. (11A) Microspeckofluorometric amlysis of celluiar monoamines after formaldehyde or glyoxylic acid condensation. Bjorkiund. Anders; Falck. Bengt; Lindvail, Olle; Methods Brain Res., 1975, 249-94. Bradley, Philip B., Ed.; Wiley: Chichester, Engl. (12A) Polarized fluorescence and Raman spectroscopy. Bower. D. I.; Struct. Prop. OrientedPolymn., 1975, 187-218. Ward, !an Macmillan, Ed.; Wiley; New York. N.Y. (13A) The luminescence assay of drugs. Bridges, James W.; Methodol. Dev. Biochem., 1976, 5, 25-38. (14A) Chemiluminescence microanalysis of substrates and enzymes. Brolin, S. E.; Wettermark, G.; Hammar, H.; Strahlentherapie, 1977, 153(2), 124-31. (15A) Protein chromophore luminescence (model studies). Burshtein, E. A : Itogi Nauki Tekh. Bioflz.. 1976, 6. 213 pp (Russ.). (16A) Archaeologicai Dating by Thermoluminescence. Cairns, T.; Anal. Chem., 1975, 4613). 266A-80A. (17A) Biochemical Fluorescence: Concepts. Vol 1 . Chen, Raymond F , Edelhoch, Harold, Ed.; Dekker. New York, N.Y., 1975, 408 pp. (18A) Biochemical Fluorescence Concepts, Vol. 2. Chen, Raymond F , Edeihoch, Harold, Ed.; M. Dekker: New York, N.Y.. 1976, 535 pp, (19A) Principles and applications of electroluminescence. Degenhardt, Heinz, Naturwissenschaften. 1976, 63(12). 544-9. (20A) Methods of surface temperature measurement using radiation. Desvignes, Francois; Rev. Gen. Therm., 1976, 15(172). 303-12 (Fr.). (21A) Fluorescent probe study of the structure of proteins. Dobretsov, G. E.; Itogi Nauki Tekh., Mol. Biol., 1975. 6. 34-104 (Russ.) (22A) Study of proteins and membranes by using fluorescent probes. Dobretsov. G. E.;Vladimirov, Yu. A.: Usp. B/ol. Khim.. 1975, 16. 115-34 (Russ.). (23A) Laser applications in medicine and biology: a bibliography. Eichler, J.; Lenz, H.; Appl. O p t , 1977, 16(1). 27-45. (24A) Optical instrumentation [for trace analysis]. Elser, R . C.; Chem. Anal. ( N .Y . ) , 1976, 46(Trace Anal.: Spectrosc Methods Elem.), 107-21. (25A) Prospects for the use of fluorescence probes in membrane studies. Fergusoii, S. J.; Struct.-Act. Relat. Chemoreception, Proc. Symp , 1975 (Pub. 1976). 25-33, Benz, G., Ed.; Inf Retr Ltd ' London, Engl. (26A) Forensic science Spectrophotofluorimetry and -phosphorimetry. Floyd, J. 6.F.: Chem Br., 1975, 11(12), 442-3. (27A) Fluorescence lifetimes of biomoiecules. Forster. Leslie S : Photochem. Photobiol., 1976. 23(6), 445-8. (28A) Use of Luminescence Spectroscopy in Clinical and Biological Analysis. Froehlich, P.; Appl Spectros. Rev., 1976, 12(1). 63-129. (29A) Luminescence of minerals in the near-infrared. Geake, J. E.; Walker, G.; Infrared Raman Spectrosc. Lunar Terr. Miner, 1975, 73-89. Karr, Clarence, Jr.. Ed.: 4cademic. New York. N Y . (30A) Review: Applications of Luminescence in Forensic Science. Gibson, E. P ; J . Forensic Scl , 1977, 22(4), 660-96. (31A) Fluorescence analysis on solid surfaces. GuilbauR. George G.; Photochem. Photobioi., 1977, 25(4), 403-1 1. (32A) Molecular fluorescence spectroscopy Guilbault, G G.; Compr. Anal. Chem., 1977, 8. 71-205. Svehla, G., Ed.; Elsevier: Amsterdam, Neth. (33A) The application of ternary complexes to spectrofluorometric analysis. Haddad, P. R . , Talanta, 1977. 24(1). 1-13. (34A) Quantum beats and time-resolved fluorescerice spectroscopy. Haroche, Serge; Top. Appl. phys., 1976, 13 (HighResolut. Laser Spectrosc.), 253-313. (35A) Bioluminescence and chemiluminescence. Hastings, J. Woodland; Wilson. Therese; Photochem. Photobiol., 1976, 23(61, 461-73. (36A) Light. Hosoya, Haruo: Kagaku Kyoiku, 1976, 24(1), 57-62 (Jpn). (37A) Biopolymer and fluorescence. Ichimura, Sachiko; Zama, Mitsuo; Kyoritsu Kagaku Raiburari, 1975. 10 iKeiko Gensho), 191-209 (Jpn). (38A) Time decay fluorometry bv DtlGtOn counting !senberg. Irvin: Biochem. Fluoresc Coricepts, 1975. 1. 43-77 Chen, Raymond F.; Edelhoch. Harold. Ed.: Dekker: New York, N.Y.
ANALYTICAL CHEMISTRY, VOL, 50, NO. 5, APRIL 1978 (39A) Bioluminescence-the biochemistry of photogens and photoagogika. Johnson, Frank H.; Shimomura. Osamu: Trends Biochem. Sci.. 1976, l(11). 250-53. (40A) Lasers: Physics, Chemistry, Biophysics; Joussot-Dubien, J,, Ed., 1975, Elsevier, Amsterdam. (41A) Fluorimetric kinetic techniques. Chemical relaxation and stopped-flow. Jovin, Thomas M.; Biochem. Fluoresc.: Concepts. 1975, 1, 305-74. Chen, Raymond F.; Edelhoch, Harold, Ed.; Dekker: New York, N.Y. (42A) The appliaticm of fluorescenceand fluorescent probes in biological systems. Kapoor, M.; Blochem. Rev., 1976, 47, 27-51. (43A) Organic fluorescence reagents for the investigationof enzymes and proteins. Kanaoka, Yuichi: Angew. Chem., 1977, 89(3), 142-52 (Ger). (44A) Fluorescence spectroscopy. Karasek, F. W.; Res.lDev.. 1976, 27(1), 28-30, 32, 34. (45A) Fluorescence and membrane potential. Kasai, Michiki; Kagaku (Kyoto), 1976, 31(7), 567-9 (Jpn). (46A) Fluorescence spectra, especially the application to membrane systems of living organisms. Kasai. Michiki; Kagaku(Kyot0) Zokan. 1977, 71. 49-66 (Jpn). (47A) Novel methods for the fluorometric analysis of proteins. Kinoshita, Toshio: Kagaku No Ryoiki, Zokan. 1976, 114, 23-30 (Jpn). (48A) An optical look at the environment. XI. The techniques of fluorescence analysis. 111. Comparison between fluorescence and other light-scattering methods of analysis. Kiainer, Stanley M.; Cody, Charles A,: Opt. Eng., 1976. 15, ( l ) , SR14-SR15. (49A) Analysis of the benzo[a]pyrene-nucleic acid complex by a fluorometric method. Kodama, Masahiko; Kagaku No Ryoiki, Zokan, 1976, 114, 47-54 (Jpn). (50A) Sensitized fluorescence and quenching. Krause, L., Adv. Chem. Phys.. 1975, 28 (Excited State Chem Phys.), 267-316. (51A) Fluorescent probes of chromosome structure. Latt, Samuel A,; Brodie, Scott; ExcitedStates Biol. Mol., Proc. Int. Conf.. 1974, (Pub 1976). 178-89 Edited by Birks, John B.; Wiiey: Chichester, England. (52A) Fluorescent nucleosides and nucleotides. Leonard, Nelson J.; Toiman, Glen L.; Ann. N . Y . Acad. Sci., 1975, 255 (Chem., B i d , Clin. Uses Nucleoside Analogs), 43-58. (53A) Microscale manipulations in chemistry. Ma, T. S . ; Horak, V.; John Wiley & Sons: Chichester
