Molecular fluorescence, phosphorescence, and chemiluminescence

Anal. Chem. 1982, 5 4 , 131 R-150R (196) Wuethrlch, K.; Nagaywna, K.; Ernst, R. R. Trends Biochem. Scl. 1979, 4 , N178-Nl81. (197) Freeman, R. Proc. R . SOC. London, Ser. A 1980, 373, 149-78. (198) Geiger, A.; HoIz, M. J . Phys. E . 1980, 73,897-707. (199) McCausland, M. A. H.; Mackenzle, I. S. Adv. Phys. 1879, 2 8 , 305-456. (200) Barnes, R. Q. “Handb. Phys. Chem. Rare Earths”; Gschneider, K. A., Jr., Eyrlng, LeRoy, Eds.; North-Holland: Amsterdam, 1979; Vol. 2, pp 387-505. (201) McGarvey, B. R. NATOAdv. Study Ins. Ser., Ser. C, 7978 1979, 44, 309-36 ---

(202) Bteaney, 8 . Bull. Ma@?.Reson. 1981, 2, 7-8. (203) Fischer, R. El. NATO A&. Study Inst. Ser., Ser. C, 7978 1979, 44, 337-77. ( 2 6 ) Luke, W. [I.; Streitwleser, A., Jr. ACS Symp. Ser. 1980, 737, 93-140. (205) Lincoln, S. F . Pure A@. Chem. 1979, 5 7 , 2059-65. (206) Tunstall, D. P. Phllos. Mag. B 1980, 42, 735-49. (207) Berthier, C.; Chabre, Y.; Segranson, P. Physlca B & C 1980, 9 9 , 107-16. (208) Lechert, H. NATOAdv. Study Inst. Ser., Ser. C 1980, 6 7 , 143-73. (209) Cabane, B. NATO Adv. Study Inst. Ser., Ser. C 1980, 67, 321-9. (210) Tabony, J. Prog. Nod. Magn. Reson. Spectrosc. 1980, 74, 1-26. (211) Resing, H. A. NATOAdv. Study Inst. Ser., Ser. C 1980, 6 7 , 219-38. (212) Fendler, E. J.; Rosenthal, S. N. NATO Adv. Study Inst. Ser.. Ser. C 1980, 61, 353-69. (213) Griffin, R. G.; Bodenhauser, G.; et al., Philos. Trans. R . SOC.London, Ser. A 1981, 209, 547-63. (214) Andrew, E. R. Phllos. Trans. R . SOC.London, Ser. A 1981, 299, 505-20. et al., Philos. Trans. R . SOC. London, (215) Schaefer, J.; Stejskall, E. 0.; Ser. A 1981, 299, 593-608. (216) Stoll, M. E. Phllos. Trans. R . SOC. London, Ser. A 1981, 299, 565-84. (217) Hahn, E. L. Faraday Symp. Chem. SOC. 1979, 73, 7-18. (218) Mansfield, P. Philos. Trans. R . SOC. London, Ser. A 1981, 299, 479-96. (219) Gray, G. A.; Hill, H. D. W. Ind. ResJDev. 1980, 22, 136-40. (220) Walker, S. M. Nucl. Magn. Reson. 1979, 0 , 200-23. (221) Walker, S. M. Nucl. Magn. Reson. 1980, 0 , 128-52. (222) Gersteln, B. C. NATO Adv. Study Inst. Ser.. Ser. C 1980, 67, 175-205. (223) Derbyshire, W. Nucl. Magn. Reson. 1980, 9 , 256-307. (224) Herzog, P. Hyperfine Interact. 1980, 8 , 215-27. (225) Wllson, G. V. H.; Chaplin, D. H. Hyperfine Interact. 1981, 10, 1081-100. (226) Stone, N. J. NATO Adv. Study Inst. Ser., Ser B . 1979, 8 4 7 , 189-204. (227) MacLaughlln, D. E. Hyperfine Interact. 1981, 8 , 749-56. (228) Alloul, H. Bul/ Magn. Reson, 1981, 2 , 2-5. (229) Turrell, B. G. Aust. J . Phys. 1979, 32,647-82.

(230) Tayal, V. P.; Srivastava, B. K.; Khandelwal, D. P.; Bist, H. D. Appl. Spectrosc. Retv. 1980, 76, 43-134. LITERATURE FOll TABLE 11

(1) Stanislawski, D. A,; Van Wazer, J. R. Anal. Chem. 1980, 52, 98-101. (2) Delpuech, J. J.; Hamza, M. A.; Serratrlce, G. J. M a p . Reson. 1979, 36, 172-0. (3) Barrett, J. L. J. Radlat Curing 1979, 8 ,20-6. (4) Rackham, D. M. Spectrosc. Lett. 1980, 73,321-7. (ti) Knoo, M.; O’tlara, K.; Shloml. Y. Antlmlcrob .-Agents Chemother., 1980, 77, 16-19. (6) Rutan, V.; Blinc, R.; Ehrenberg, L. J. Magn. Reson. 1980, 40, 225-7. (7) Ho, F. F. L.; i(losiewicz, D. W. Anal. Chem. 1980, 5 2 , 931-16. (8) Tiwarl, P. N.; Burk, W. J . Am. OilChem. SOC.1980, 5 7 , 19-21. (9) Mossa, J. S.; El-Obeiid, H. A,; Hassan, M. M. A. Spectrosc. Lett. 1980, 73, 149-57. (110) Tulloch, A. P.; Bergter, L. Lipids 1979, 74, 996-1002. (111) Hassan, S. !3. M. Methods Enzymol. 1980, 6 7 , 552-6. (112) Iida, T.; Jeong, T. M.; Tamura, T.; Matsumoto, T. Lipids 1980, 75, 86-8. (113) AI-Badr, A. A.; Ibrahim, S. W. Spectrosc. Lett. 1980, 73, 143-50. (14) AbouCEneln, H. Y. J. Pharm. Pharmacol. 1979, 3 7 , 196. (15) Avdovich, H W.; Neville, G. A. Can. J. Pharm. Sci. 1981, 75,75-7. (18) Neumann, H.; Vordemaier, G., Arch. Klrminol. 1981, 787, 33-42. (17) Knoll, J. A. J . Forensic Sci. 1979, 2 4 , 303-6. (18) Aboutabl, E. A,; Mossa, J. S., Hassan, M. M. A. Spectrosc. Lett. 1979, 72,579-90. (19) AI-Badr, A. A.; Ibrahlm, S. E. Spectrosc. Lett. 1979, 72,419-26. (1’0) Loutfy, M. P,.; Hassan, M. M. A. Ibid. 1979, 72,591-601. (21) AbouCEnein, H. Y.; ACRashocd, K. A.; El-Fatatry, H. M. Chem., Biomed. Envlron. Instrum. 1980, 70 237-47. (22) Aboutabl, E. A.; El-Fatatry, H. M. Pharmazie 1980, 3 5 , 231-2. (23) Aboutabl, E. A,; Hassan, M. M. A. Talanta 1980, 27, 679-81. (24) Hassan, M. IW. A,; Jado, A,; Loutfy, M. A. Spectrosc. Lett. 1980, 73, 595-602. (25) Krivdin, L. B ; Chekareva, T. G.; Sakharovskii, V. G.; Romanova, 1. B. Zh. Anal. Khim. 1981, 3 6 , 357-63. Chem. Abstr. 1981, 9 4 , 154880q. (26) Hassan, M. M. A,; Aboutabl, E. A. Spectrosc. Lett. 1979, 72,3 5 1 4 3 . (27) El-Obeid, H. A.; Hassan, M. M. A. Spectrosc. Lett. 1979, 72,555-7. (28) Visintalner, ,J.; Beebe; D. H.; Myers, J. W.; Hirst, R. C. Anal. Chem. 1981, 5 3 , 1570-2. (29) Giliet, S.; Rutilni, P.; et al. Fuel 1981, 60, 221-5, 226-30; Srivastava, S. P.; Singh, I. D. J. Chem. Techno/. Blotechnol. 1980, 30, 727-30. (30) Monk, W. B ; Poranski, C. F.. Jr. Org. Coat. Plast. Chem. 1978, 39, 99-102. (31)-SojG, S. A.; Wolfe, R. A. Appl. Spectrosc. 1980, 34, 90-3. (32) Silvelra, A., h‘.; Bretherick, H. D., Jr.; Negishl, E. J. Chem. Educ. 1979, 56, 560. (33) Barron, P. F.; Wilson, M. A,; Stephens, J. F.; Cornell, B. A.; Tate, K. R. Nature (Londor,’)1980, 286, 585-7. I

Molecular Fluorescence, Phosphorescence, and Chemiluminescence Spectrometry E. L. Wehry Department of Chemistry, University of Tennessee, Knoxville, Tennessee 37996

As in the previous review in this series (AI), this survey stresses advances in the techniques of luminescence spectrometry and in instrumentation related to present or potential analytical lumiinescence methods. Applications are cited only when they seem particularly novel to this reviewer. The review, prepared with the assistance of a computer search profile of Chemical Ab:rtracts titles and identifiers prepared locally, covers literature indexed by Chemical Abstracts from December 1979 Wol. 91, issue 23) through October 1981 (Vol. 95, issue 20). Many journals scanned manually by the author are covered up through issues received by November 30,1981. As in the previous review, certain topics are excluded; for example, virtually all publications concerning atomic fluorescence, moleculiilr luminescence in flames, X-ray fluorescence, solid-state phosphor and semiconductor luminescence (both organic and inorganic), radioluminescence, liquid scintillation counting, and photosynthesis and solar energy conversion have been excluded. Papers dealing with 0003-2700/82/ 0354- 13 1 R$OS.OO/O

luminescence detection in liquid, thin-layer, or paper chromatography ai*e cited only when they appear to be of uppreciable spectroscopic interest. The immense literature on fluorescent probing of macromolecular and micellar systems, much of which could quite properly be subsumed in the rubric of‘ “analytical chemistry”, has been excluded almost totally except for citation of a few general reviews. Certain other subject matter areas, noted in the appropriate sections of the review, are covered in a highly arbitrary manner. The literature related to luminescence analysis continues to expand rapidly. For example, I estimate that 14% of the full papers published in Analytical Chemistry in 1981 could have qualified for inclusion in this review on the basis of their subject-matter coverage! Thus, to keep the number of references and length of the review from becoming unduly preposterous, a great deal of arbitrary exclusion of interesting work was necessary. I apologize to those authors whose work has been slighted. 0 1982 Amerlcan Chemical Society

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One change in format from the preceding review ( A I ) : generally, methodology and applications of a particular technique are here discussed together, rather than in separate categories as was done previously.

GENERAL BOOKS AND REVIEWS The second edition of the “Treatise on Analytical Chemistry” incorporates a broad survey of fluorescence and phosphorescence fundamentals, instrumentation and techniques, and a plications by Seitz (BI). Penzer has prepared a very readagle general review of fluorescence and phosphorescence, with emphasis on analytical applications and the use of fluorescent probes in biological systems (B2). Wehry had edited two additional volumes of the “Modern Fluorescence Spectroscopy” series (B3);the individual chapters are cited in the appropriate sections of this review. Volume 10 of the Chemical Society’s “Specialist Periodical Report” series on Photochemistry includes a 114-page chapter on ”Developments in Instrumentation and Techniques” (B4)and a 54-page chapter on “Photophysical Processes in Condensed Phases” (B5);both of these superb surveys contain much material of direct analytical relevance. Bell has reviewed the general principles of molecular fluorescence in liquid solution media and has discussed (in general terms) the biochemical applications of solution luminescence (B6). Snell has surveyed fluorometric and absorptiometric methods for determination of nonmetals (B7). Seitz has reviewed the variety of procedures which can be used to convert nonluminescent analytes into products detectable by fluorescence or phosphorescence. Applications to both organic and inorganic analyses are cited; many examples are provided, and nearly 300 references are listed (B8). A new book (in German) by Zander stresses practical aspects of fluorometric analysis (B9). An edited compilation of chapters on ”Lasers in Chemical Analysis” includes a number of chapters pertaining to applications of lasers in fluorometric analysis (BIO). A review of molecular fluorescence induced by electron impact surveys the literature and briefly discusses potential analytical applications (B11).A monograph on radiationless transitions edited by Lin is of considerable fundamental interest (B12). The luminescence kinetics of transition-metal complexes in liquid solution have been reviewed in great detail by Kemp (B13). Other books and reviews, dealing with more specific topics, are cited in subsequent sections. INSTRUMENTS AND INSTRUMENTAL COMPONENTS Lamps and Other Nonlaser Sources. A surface spark discharge flash lamp, which emits over the 270-390 nm range, may have utility in molecular photoluminescence spectrometry (C1). A somewhat unconventional and not inexpensive (but very intense) source for molecular fluorometry is an inductively coupled plasma discharge, with the output wavelengths of course depending upon the element(s) aspirated into the ICP (C2). The characteristics of synchrotrons and synchrotron radiation, and the characteristics of the various facilities having storage rings, are discussed by Winick (C3). Lasers. As laser technology continues to improve, lasers will inevitably experience increasing use in analytical photoluminescence spectrometry, and a fairly thorough coverage of developments in laser science (with emphasis on tunable dye lasers) therefore is warranted. Several reuiews are of interest. Tang has presented a useful introductory discussion of basic laser concepts (C4). Wright and Wirth have provided a very readable summary of the fundamentals of laser action and the characteristics of the common types of lasers (C5). Lytle has discussed laser fundamentals in considerabledetail, with emphasis on optical amplification phenomena, pumping schemes, and the behavior of optical resonators (C6). The operating characteristics of pulsed lasers have been surveyed by Harris (C7). Volume 3 of the “Laser Handbook includes chapters on second harmonic generation and ultrashort pulse generation, as well as surveys of dye, excimer, and gas lasers (C8). A handbook dealing with the optical hazards of lasers (and other very bright light sources) will be useful to individuals planning initial entries into the area of laser fluorometry ((29). 132R

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Tunable lasers are of special interest for luminescence measurements. Wirth has reviewed the operating principles of tunable laser systems (C10).A readable short summary of recent developments in tunable lasers (dye, excimer and exciplex, and free-electron lasers) has been presented by Pidgeon and Colles (C11). Peterson has reviewed dye lasers in depth (C12). A useful tabulation of structural formulas and synonyms for the names of 30 common laser dyes has appeared (C13). The wavelength tuning ranges and optimum solvents and concentrations are reported for 16 commercial dyes, using the third harmonic of a Nd:YAG laser (355 nm) as pump (C14). Photodecomposition of dyes can be a major headache when high-power excimer lasers are used for pumping; a study of the effects of various solvents upon the stability and average power obtainable from XeC1-pumped dyes is of considerable value (‘215). Incorporation of triplet-quenching functional groups as structural entities in dye molecules is reported to produce much more efficient (Le., intramolecular) triplet quenching than is achieved by the procedure of adding triplet quenchers to conventional dye solutions in flashlamp-pumped dye lasers (C16). A common problem in using a dye laser is what to do when the desired wavelength is nowhere near the gain maximum for any dye. An obvious ploy is to try a mixture of dyes. This approach can indeed work, especially for cases in which the output of the lasing dye is normally attenuated by self-absorption, which is a particularly serious problem in transversely pumped dye lasers (C17). Determining the actual wavelength and spectral band-pass of the output of a dye laser is not necessarily a trivial undertaking. A home-built monochromator system coupled to a diode array detector has been designed for such measurementa (C18). A procedure for accurate calibration of the lasing wavelength of a dye laser fitted with an intracacity etalon has been described ((719). The design of a three-stage birefringent filter, reportedly smoothly tunable over the 440-660 nm range, has been described (C20). A useful, detailed discussion of the problems which must be overcome to construct an automatically scanning dye laser containing more than one tuning element has appeared (C21). A dye laser is sensitive to small optical gains within the laser cavity, and this fact can be used to lock a large portion of the laser output onto wavelengths correspondin to the fluorescence spectrum of a fluorescent sample placetinside the cavity. Characteristics and potential applications of this “intracavity gain probing”technique have been discussed (C22). The peak power of a Hansch-type nitrogen-pumped dye laser reportedly increases significantly when the dye solution is cooled (C23). N2-pumpeddye lasers which employ a grating operated at grazing incidence for beam expansion (instead of a telescope, as in the conventional Hansch design) have been designed (C24-CZ6). A similar approach for obtaining highenergy spectrally narrow output from a flashlamp-pumped dye laser has been reported (C27). Optimum concentrations, efficiencies, solvents, and tuning ranges for a large number of dyes pumped by an XeCl excimer laser (308 nm) have been compiled (C28). Pulse energies on the order of 1 J (!) have been produced from dyes pumped by a 4-5 XeCl excimer laser (C29). A new dye cell design (basically a prism with a hole drilled in it, through which the dye circulates) is reported to alleviate optical cell damage problems and the relatively low beam quality characteristic of excimer-pumped dye lasers (C30). Intracavity doubling of a ring dye laser has been accomplished, and the desirable characteristics of such a configuration for high-resolution spectroscopy in the UV have been discussed (C31-C34). A method for rapid scanning of commercial ring dye lasers has been described (C35). An atlas of wavelengths available from gas lasers has appeared; 6145 different lines can be obtained in the 5086000-~m-~ range (C36). Two reviews of gas and ion lasers are of interest ((737, C38). The principles and operating characteristics of excimer lasers have been reviewed by Hutchinson (C39). Various procedures for extending the lifetime of gas mixtures in excimer lasers have been suggested (C40, C41). Production of high-power, high-purity light at 248 nm by using a KrF laser as an amplifier of frequencydoubled radiation from an excimer-pumped dye laser has been reported, and use of this system for two-photon-induced


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specuoscopy. trace mganic analysis by spectroscopic methods. low-temperature techniqws in analytical spectroscopy. amlyticai BPPliCB~"S 01 laser SpaCbOSCOpy. and the chemical degradation of environmentai wiimnh. tk is ednw 01me m w r a p h Mddsrn Fhmescenn, Spectmsmpy and currently wwes on the ednaial h r d S 01 A M m I Lettm and mS mOmgraph wries Contemporary Insmrmntetion and AnsWsIs He is B member of the American Chemical Society. Soclety faApplied SpCtrOscopy, Optical Sociely of America. A m BriCan AsroClatiMI la Me Advancement Of Science. and Inter-Amsrlcan PhOtocbrnlcal society

fluorescence studies is discussed (C42). A stepwise procedure for refilling the reservoir of a commercial argon ion laser, without removing the plasma tube from ita mount (thereby reducing the probability of breaking the tube), has been described (C43). An inexpensive system for coolin an Ar+ laser with deionized water has been described (844). Noise power spectra for a number of commercial Art lasers have been published (45). Harmonic generation, s u m and difference frequency generation, and other nonlinear optical phenomena are surveyed in a very readable manner by Wright (C46). Stimulated Raman scattering, which can be used to generate tunable output both in the UV and near-IR (where dyes either do not exist or have unsatisfactory performance parameters), is the subject of a brief review by Byer (C47). Many UV wavelength can be generated by stimulated Raman scattering in H,or 0, pumped by a NdYAG laser or harmonics thereof (C48). Compensation for pulse-bpulse amplitude variations, and other noise sources, is a perennial problem with pulsed lasers. A digital averaging circuit for accomplishing this purpose, used in conjunction with a sampling oscilloscope, has been described (C49). Measurement of the pulse energy of a N, laser with the ferrioxalate (C50)or azobenzene (C51) chemical actinometers has been described. Applications of lasers to analytical molecular fluorescence spectrometry are increasing rapidly in number, analytical relevance, and practicality. A useful overview of the use of lasers in analytical spectroscopyhas been preaented by Wright and Wirth (C52). Richardson has prepared a short survey (C53)and a bwk chapter (C54) dealing with applications of lasers in fluorometry. Wright has summarized the w e of lasers in fluorescence spectroscopy (C55). The use of lasers to achieve greatly increased selectiuity in fluorometric analysis continues to expand. Wright and *workers have reviewed the determination of trace quantities of lanthanide ions by selective laser excitation of 'probe ion luminescence" in ionic solids (C56C58). Modifications of the basic probe ion luminescence method to increase quantitative precision and decrease the effects of interfering ions have been reported. Determination of nonfluorescent ions in CaF, precipitates is achieved by clustering with a luminescent probe ion such as Er3+; the luminescence excitation and emission spectra of the probe ion are shifted by this defect cluster formation, and laser 'site-selective" excitation of fluorescence from probe ions clustered with specific analyk ions can be effected. Energy-transfer phenomena in such systems also may have analytical utility (C59). A method for determination of uranium in aquatic samples proceeds via coprecipitation of UOZ2+in CaF, followed by dye or Ar+ laser excitation of U06%luminescence in the calcined precipitate (C60, C62). The advantages of using laser excitation to achieve selective excitation of fluorescence from individual constituents of complex organic mixtures by various types of low-temperature luminescencetechniques have received considerable attention (C62-C69). Interest in the use of lasers to achieve improved detection limits in fluorometric analysis remains intense. Yeun and Sepaniak have discussed the advantages of laser-injuced

fluorescence detection in liquid chromatography (C70, C71). Zare and eo-workers have described the application of laser fluorometry to enzyme immunoassay (C72, C73). Laser excitation has been reported to produce generally better detection limits than lamp excitation in drug analysis (C74,C75). A detection limit of 2 pg/L is reported for N,-laser excited fluorometric determination of Eu(II1) as the HTTA chelate (C76). A European commercial fluorometer, which uses a N laser as source, has been used to determine uranium as U0,21 in natural water samples with a reported detection limit of 0.05 ng/mL (C77). The use of lasers also enables the development and application of new spectroscopic measurements not feasible with conventional sources. Kaiser and co-workers have described a two-photon fluorescence measurement wherein an infrared laser pulse of several picoseconds duration produces vibrationally excited molecules in the ground electronicstate, which are then promoted to vibronic states of the lowest allowed electronically excited state by a second picosecond pulse (this time in the UV). Fluorescence excitation spectra obtained in this way for large molecules in liquid solution contain much more structure than those obtained by conventional onephoton excitation, a fact which may have substantial analytical importance (C78). A further analysis of such doubleresonance experiments by Wright indicates some of the analytical possibilities and also considers possible interference in the measurements from other phenomena (such as CARS) (C79). Wirth and co-workers have reviewed the principles and experimental techniques of 'two- hoton polarization spectroscopy" (C80) and have consigred the feasibility of employing polarization-dependent two-photon fluorescence excitation spectra for identifying isomeric compounds in mixtures (C81). Richardson and co-workers have reported a two-laser fluorescence experiment, wherein a CW UV or visible laser and a pulsed or chopped IR laser beam simultaneously impinge upon the same volume region of a sample. A modulated fluorescence signal results, which is claimed to arise from temperature effects upon the fluorescence quantum efficiency of the analyte, rather than from a double-resonance phenomenon. Selectivity is attained by choosing the IR laser frequency such that only the desired sample constituent absorbs at that frequency (if possible) (C82). The ability of metal ions to "quench" emission from dye lasers, when added directly to the dye solution, appears in some cases to result from true quenching of the dye emission rather than from intracavity absorption phenomena. The observation may lead to sensitive methods for metal-ion determinations (C83). The literature currently is replete with reports of the use of a laser besm to photoehemidy fragment molecules in the gas phase, following which fluorescence is excited from one or more of the fragments with a second laser beam. A thwretical paper discusses the potential applications of such phenomena to analytical chemistry in great detail, though no actual analytical data are presented (C84). A fluorescence method for isotopically selective determination of la12 employs a broad-band dye laser filtered by an intracavity cell containing ln12 at sufficient ressure to quench laser output a t all wavelengths a t which ?71, absorbs (C85). Remote sensing applications of laser-induced fluorescence continue to attract attention. A monograph on optical techniques for remote sensing from aircraft and satellites has appeared (C86). The advantages of fluorescence over other "LIDAR" processes have been discussed, and equations for SJN optimization in fluorescence LIDAR have been presented ( 87) Potential application of the laser-induced fluorescence of UOZ2+for remote sensing of uranium in geological samples has been discussed in detail (C88, C89). A frequency-doubled NdYAG laser LIDAR system for simultaneous measurement of water Raman backscatter and fluorescence by chlorophyll (and other naturally occurring aquatic fluorophores) has been described (C90).The Raman backscatter signal (0-H stretch) is used to normalize for variations of the penetration depth of the laser beam into the body of water as a function of the optical attenuation properties of the surface (C90-C92). I t is worth noting that dissolved humic materials in lake waters may fluoresce efficiently, and fluorescence signals therefrom may often by assigned incorrectly to chlorophyll (C93). A tunable dye laser fluorescence LIDAR system, installed in a helicopter, is used to detect fluorophores in ocean waters (C94). An airborne ANALYTICAL CHEMISTRY, VOL. 54. NO. 5, APRIL 1982

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instrument for oil-spill detection functions via N2laser induced fluorescence (C95). A detailed description of an airborne NdYAG pumped dye laser fluorescence spectrometer for tropospheric OH radical determinations has appeared (C96). Various limitations of laser fluorescence atmospheric OH radical monitoring (including photolytic production of OH, interferent luminescence, and saturation effects) have been discussed, and various remedies have been suggested (C97-C101). Monochromators. The principles of grating and prism Monochromators have been reviewed by Meehan (C102). Techniques for aligning Czerny-Turner and Ebert-Fastie gratin monochromators have been described (C103). A procecfure for accurate three-dimensional alignment of a monochromator has been described (C104). Mathematical formulas for calculating the dispersion of a Czerny-Turner monochromator are relevant to use of this common configuration with electronic array detectors (C105). A “bolt-on” computer-controlled stepping-motor drive, which reportedly can be mated to commercial monochromators without major renovation, has been described (C106). Recipes for liquidsolution filters prepared from aqueous solutions of transition-metal salts have been published (C107). Sample Illumination Optics. A fluorometer which automatically corrects measured fluorescence intensities for absorption of incident and emitted radiation by the sample has been described. This instrument functions by performing spectral measurements at several different sample cell positions, chosen such that the effective optical pathlengths for excitation and fluorescence are varied. It is reported to produce linear analytical calibration curves, even for very strongly absorbing samples (C108, C109). Optical components for converting a commercial singlebeam absorption spectrophotometer into a fluorometer for either front-surface or right-angle illumination have been described (C110). Conversion of a commercial dual-wavelength filter photometer into an automated ratio-correcting filter fluorometer employing straight-through illuminationhas been described (C111). The use of front-surface illumination for determination of bilirubin and fluorescent proteins in undiluted blood samples, without extraction, is discussed (C112). A “hematofluorometer”is a front-surface fluorometer used to determine porphyrins (e.g., zinc protoporphyrin) in whole blood. Techniques for overcoming interference from nonspecific fluorescence of plasma in such measurementshave been discussed (C113). The use of such a front-surface instrument to determine hemoglobin in blood, via inner-filter absorption by hemoglobin of the fluorescence of a dye added to the blood sample, has been reported ((7114). Modificationsof a commercial fluorometer for illuminating samples with a laser beam have been described in detail (C74). Investigators contemplating the use of high-power UV lasers will find it useful, though not necessarily enjoyable, to note that quartz, sapphire, MgF2, and BaF, all fluoresce under high-power UV illumination (C115). Choices of materials for fabrication of sample containers and other optical elements, and design of illuminationand collection geometries, may thus be crucial. Much attention has been devoted to the fluorometric examination of very small and/or inaccessible samples. Techniques for fluorescence measurements of samples having volumes on the order of picoliters have been described in detail (C116). Microcuvettes,having volumes in the 8-80 pL range, suitable for fluorescence measurements have been described (C117). The design and characteristics of disposable miniature plastic reaction chambers, having volumes in the 5-10 p L range, have been discussed with regard to fluorometric enzyme determinations (C118). Use of a random bifurcated fiber optic bundle for measurement of luminescence from hazardous or inaccessible samples has been discussed (C119). A computer-controlled microfluorometer for detecting organic particulate contaminants on solid surfaces has been described (C120). Procedures for calibrating microspectrofluorometric instrumentation to produce corrected emission spectra have been described (C121). Techniquesfor two-dimensional microfluorometric scanning of nerve fibers, at a resolution of 0.5 pL, have been reported (CI22). Fiber optic techniques for simultaneous visual examination (i.e., endoscopy) and electronic detection of fluorescence (using fluorescent marker compounds) in the 134R


human respiratory tract have been described (C123). The microfluorometric examination of biological cells requires solving a host of technical problems; progress in this area is discussed in a subsequent section of this review. Detectors. A useful overview of the various types of detectors of UV and visible radiation, and of noise sources associated with detection, has appeared (C124). The operating principles of the photomultiplier tube (PMT) have been reviewed (C125). A “PhotomultiplierHandbook”contains much practical information of value to users of PMTs (C126). Meade has presented an extremely helpful review of photon counting, with special emphasis on desirable characteristics of PMTs and ancillary electronics,the origin of spurious PMT pulses, and optimization of the operating parameters of photon-countingsystems (C127). The use of photon counting for detection of weak optical signals has been reviewed by Seliger (C128). A computerized system for changing both the anode current and dynode voltage of a PMT has been described; use of such a system is claimed to extend the linear dynamic range of the tube to as much as 7 decades in incident optical power (C129). Detailed descriptions of two microprocessor-controlledwide dynamic range integrating readout systems for PMTs have appeared (C130). A modular photometer which permits rapid switching between dc and photon-counting modes (C131) may have useful applications in luminescence measurement. Enke and co-workers have discussed in detail the factors influencing the relative efficiency of pulse-counting experiments;particular emphasis is devoted to adjustment of the integration time to achieve optimal efficiency (C132). Detailed measurements and discussion pertaining to the dependence of PMT gain on count rate have been presented (C133). A circuit for measurement of gain changes in PMTs is useful for making corrections for these effects in photon-counting experiments (C134). A high-speed amplifier-discriminatorand counter for photon counting has been designed (C135). Christian, Callis, and Davidson have reviewed the operating characteristics of electronic array detectors, and their use in the fluorometric analysis of mixtures, in depth (C136). The use of extended-delaytarget integration techni ues for signal averagin of weak optical signals using a coolej SIT uidicon tube hasteen described (C137). A differential voltage readout technique,which reportedly increases the linear dynamic range of a SIT vidicon in experiments involving use of pulsed sources, has been described (C138). Interfacing of a SIT vidicon spectrograph to a CAMAC-based minicomputer has been described (C139). The operating principles of photodiode arrays and charge-coupled devices have been reviewed by Hall (C140). Talmi and Simpson have discussed the characteristics of a 1024-element diode array in considerabledetail (C141). Ingle and Ryan have discussed the characteristics of an intensified diode array and its application to fluorometric rate methods of analysis (C142). A microprocessor-controlled photodiode-array spectrograph for measuring fast chemiluminescence spectra has been described (C143). A monograph on charge-coupled devices includes much material of both fundamental and practical interest (C144). The principles and operating characteristics of image intensifiers and microchannel plates have been reviewed (C145). A detailed discussion of the operating principles and characteristics of microchannel plates employing curved channels has appeared, and the use of these devices in the pulse-counting mode has been discussed (C146). Whenever an array detector of any type is used, it is desirable to be able to calculate the dispersion of the monochromator to which it is attached. Mathematical expressions for such computations applicable to the common CzernyTurner grating monochromator configuration have appeared (C105). Computerized Instruments, Auto-Analyzers, etc. A microcomputer-controlled fluorometer which produces corrected spectra, first derivative spectra, and fluorescence quantum yields has been described (‘2147). A microprocessor-controlled fluorometer for use in fluoroimmunoassays has been described (C163). A computer-controlled pulsed dye laser fluorometer, wherein both scanning of the dye laser and synchronization of data acquisition with the dye laser wavelength setting are controlled by the computer, has been described, Such an instrument is well suited to signal averaging

MOLECULAR FLUORESCENCE. PHOSPHORESCENCE, AND CHEMILUMINESCENCE SPECTROMETRY

in spectral acquisition, which is traditionally a somewhat difficult operation when a scanning pulsed dye laser is used as source (C148). The published proceedings of a symposium on centrifugal analyzers in clinical chLemistry include reviews on fundamental concepts of centrifugal analyzers (C149) and techniques and applications of fluorescence measurements made with centrifugal fast analyzers (C150) by Tiffany and co-workers. The use of a commercial centrifugal analyzer outfitted for fluorescence measurements for kinetic fluorometric enzyme determinations has been described (C151). A portable fast centrifugal analyzer has been employed for kinetic analyses via chemiluminescence measurements (C152). The design of a “fluoristat”,for use with systems in which the analyte reacts with a fluorescent substrate to form nonfluorescent products, has been described. In this device, the fluorescent reagent is added at a rate such that its fluorescence signal remains unchainged during the course of the reaction. The added quantity of reagent required to maintain constancy of fluorescence intensity can be related to the quantity of analyte present; in effect, this approach is the analogue of acid-base titrations performed with EL pH-stat. The device also can be used with reactions forming fluorescent products, in which case the “constant fluorescence intensity” condition is maintained by addition of a fluorescencequencher. In the latter case, however, ithe calculations are messier and linear calibration curves may be difficult to obtain (C153). Various aspects of flow-injection analysis have been reviewed by Ruz,ickaand Hansen (C154), Ranger (C155),and Pardue and Fields (C156). (Parenthetically, use of the acronym “FIA” for both “flow injection analysis” and “fluoroimmunoassay” is becoming irksome; it appears that the practitioners of fluoroimmunoassay got there first.) Experimental techniqueti for fluorometric analyses based on the flow-injection principle have been described (C157-C159). A laser fluorometer for use in conjunction with flow injection analysis, which uses a sheath flow cell, has been designed (C160). Automation of homogeneous energy-transfer fluoroimmunoassays by flow-injection techniques ([FIA],?)has been described (C161). A fluorometric method for gallium, involving a micro solveint extraction-flow injection procedure, has been described, and transient phenomena in the extraction coil have been studied by laser fluorometry (C162). Apparatus and procedures for a continuous-flowenzymatic determination of galactose in blood have been described (C164). Other Instruments. A portable fluorescencemonitor for rapid detection of poZycyclic aromatic hydrocarbons in coal conversion and oil shale plants has been developed (C165). SPECTRAL CORRECTION, COMPUTER RETRIEVAL OF SPECTRA, QUANTUM YIELD DETERMINATIONS, S / N CONSIDERATIONS, A N D RELATED MATTERS Velapoldi and Mielenz have provided a detailed description of the quinine sulfate dihydrate fluorescence standard reference material developed at the National Bureau of Standards (01).Calibration procedures for microfluorometric instrumentation have been developed (C121). Many self-correcting fluorometers employ a split-beam arrangement, wherein a portion of tlhe exciting light is split off and directed to a calibrated detector or quantum counter. Errors in this process resulting from wavelength dependences of the beamsplitter reflectance and the sample cell window transmittance have been discussed in detail (02). Mathematical procedures to correct for absorption of fluorescence in right-angle fluorometers have been described and advocated (03). An experimental technique for correcting measured fluorescence signals for absorption of incident light and/or fluorescence involves measuring the fluorescence intensity at different positions of the sample cell (C108, C109). Equations to calculate the effect of radiative energy transfer upon the fluorescence intensity of both the emitting and absorbing species have been presented (04). Luminescent quantum counters consisting of laser dyes dispersed in organic polymer films have been developed (05); these systems should prove useful for (among other purposes) fluorometer calibration. A D, lamp with a MgF, window has been proposed as a “standard lamp” in t!he 115-370 nm region (D6). In the comparative method for fluorescence quantum yield (@F) measurement, it has been contended that the “n2”re-

fractive-index correction customarily employed is not generally valid. It is argued that, if the width and height of the slit which views the luminescence are small compared with the dimensions of the fluorescing area of the sample, no refractive-index correction is needed. However, if this condition is not satisfied, no general correction procedure which accurately compensates for refractive-index errors exists (07). ]Errors in the comparative method for @F measurement produced by failure to correct for the finite spectral bandwidth of the exciting light have been discussed (08). It has been contended that iipproximation of the area under the fluorescence spectra of the unknown and standard (by using only the peak height and half-width) produces results of acceptable accuracy in the comparative @F technique (09). Mathematical techniques to correct for radiative energy transfer, and their use in aFmeasurements, have been presented (010). A detailed description of a microcomputercontrolled fluorometer,which generates corrected spectra and (PFvalues (by the comparative method) has appeared (C147). Kirkbright and co-workershave discussed the use of photoacoustic spectrometry to obtain absolute luminescence quantum yields for solid organic phosphors (011, 012). A photoacoustic technique for aFdetermination, in which the phase of the photoacoustic signal is measured as a function of the chopping frequency, has been described (013). Calorimetric procedures for measurement of absolute @F values have been described (014). Use of Raman scattering of the solvent as an internal standard in the measurement of very small @F values has been outlined (015). Faulkner and co-workers have discussed the general problem of interpreting fluorescence spectral data in terms of inolecular structure, with emphasis on computer techniques (especially file searching) for identification of individual fluorophores in mixtures (017). A general (albeit detailed and lengthy) review on computer retrieval of spectral data treats such topics as correction of spectral artifacts introduced by spectroscopic instrumentation and processing of spectral data for elucidation of molecular structure (018). The sensitivity of fluorescence spectra to such environmental parameters as solvent polarity and pH is a nuisance for compilation of liloraries of fluorescence spectra for subsequent computer searches. The possibility that computer techniques can be used to predict peak shifts and aF changes as a function of ciolvent, thereby minimizing the number of fluorescence spectra of any particular compound that would have to be incorporated into spectral files, has been discussed (019). The computer techniques of “principal component analysis” and “decompositionanalysis” have been applied to identification of individual fluorophores in two- and three-component imixtures in liquid solutions, and problems caused by similarities in the spectra of individual sample constitutents have lbeen discussed (020). A digitized fluorescence spectrum containing n points may ]be regarded as a vector in n-dimensional space. This concept has been applied to a mathematical technique for identification of oil-spill samples, by comparing the spectra of an unknown sample with those of library spectra of unweathered and laboratory-weathered oils. The vector-analysis method is used to ascertain which of the library spectra is the closest match to the unknown (021). Simplex optimization techniques have been applied to quantitative fluorometric analyses (022). Computer techniques are of course integral to use of data in the form of “excitation-emission matrices” (cf. the following section).

SYNCHRONOUS LUMINESCENCE, EXCITATION-EMISSION MATRICES, AND SPATIALLY RESOLVED LUMINESCENCE Reviews. Substantial activity continues in the development and application of techniqueswhich provide some combination of the information contained in conventional excitation and emission spectra and/or which provide information on the spatial distribution of luminescence from a sample. The principles, experimental techniques, and analytical applications of synchronously excited luminescence spectrometry have been reviewed by Vo-Dinh ( E l ) . The fundamental properties of the isxcitation-emissionmatrix (EEM), techniques for its measurement, and applications of the EEM to analysis of mixtures have been reviewed in detail by Christian, Callis, and Davidson (C136). ANALYTICAL CHEMISTRY, VOL. 54, NO. 5, APRIL 1982

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Instrumentation and Techniques. A SIT vidicon system capable of obtaining fluorescence, excitation, and coherent anti-Stokes Raman spectra of the effluent from a liquid chromatographic column has been described (E2).Use of a SIT vidicon spectrograph to generate EEMs of components eluting from a liquid chromatographic column has been deand mathematical techniques for quantitation scribed (E3), of individual components in mixtures from such data have been reported (E4). Optical systems, based on a SIT vidicon, for acquiring spatially resolved fluorescence spectra of luminescent spots on thin-layer chromatography plates have been described (E5). The design of a vidicon-based fluorometer for studies of single biological cells is described in detail by Kohen and co-workers (E6).A computerized “video microscope”, useful for spatially resolved bioluminescence measurements and fluorescence microscopy under low-intensity illumination (among other applications),uses a microchannel plate image intensifier: SIT vidicon optical system (En. Applications. Vo-Dinh and co-workers have reported on the use of synchronous fluorescence techniques for identification and quantitation of polycyclic aromatic hydrocarbons in an airborne particulate matter sample from an unnamed industrial environment (E8)and in solvent-refined coal without prior separation (E9).The use of synchronous luminescence and EEM methods in oil identification has been discussed in detail by Eastwood (EIO)and Lloyd ( E l l ) . Use of synchronous fluorescence following liquid chromatography for identification of polycyclic aromatic hydrocarbons in and coal-derived liquids has been reported (E12).Lloyd (E13) Winefordner et al. (El4) have exchanged comments regarding the utility of synchronous luminescence spectroscopy in the quantitative analysis of complex mixtures of unknown composition; the influence of inner-filter effects on quantitative reliability for such samples may be crucial. Corrections for inner-filter effects in synchronous luminescence spectrometry have been discussed ( E l l ) . The use of an intensified diode-array fluorometer for rapid sequential determination of two analytes, and for detecting spectral interferences in complex samples, has been discussed (El5). The use of EEMs in liquid chromatographic detection has been discussed (E3).The EEM method can be applied in phosphorescence,as well as fluorescence; phosphorescence EEMs have been used in oil identification (E16).Fogarty and Warner have described an ingenious use of an array-detector fluorometer for initiating and following the course of photochemical reactions in liquid solution; the generality of the technique is, however, limited by the requirement that either the reactant or one of the products be a fluorescent species (EI7). The use of EEMs for identification of fluorescent microorganisms has been discussed (E18-E20). Computational methods for making effective use of the information content of an EEM continue to be developed. A ratio method for unraveling EEMs of multicomponent samples has been discussed (E21).The use of “rank annihilation” computational procedures in the quantitative determination of individual fluorophores in complex mixtures has been discussed (E22). “Simultaneous multicomponent rank annihilation” is a mathematical procedure for computation of the concentrations of individual components in mixtures from EEM data (E23). LUMINESCENCE IN CHROMATOGRAPHIC

DETECTION The literature pertaining to use of fluorescence and chemiluminescence in chromatographic detection (especially for liquid chromatography)is enormous, and only those relatively few publications which appear to be of very general interest or applicability (or unusual apparent novelty) can be cited. Rhys-Williams has reviewed the basic concepts and applications of fluorescence detection in high-performance liquid chromatography (HPLC) (F1). The use of fluorescence for detection in HPLC and gas chromatography has been reviewed by Froehlich and Wehry (F2). Faulkner and co-workers have described in detail a computerized fluorometer for detection in HPLC and have discussed use of this instrument in conjunction with file-searching techniques for identification of specific compounds in mixtures (DI7). A readable overview of laser-induced fluorometric detection techniques in HPLC has been presented by Yeung 136R


and Sepaniak (C70). Fluorometric detection in HPLC using a SIT vidicon detector to generate excitation-emission matrices has been discussed, and a sheath flow cell suitable for use with such a detection system has been described (E3).Mathematical techniques for quantitating individual components in mixtures separated by HPLC and detected by an arraydetector fluorometer are set forth (E4). A computerized SIT vidicon system capable of obtaining fluorescence, excitation, and coherent anti-Stokes Raman spectra of HPLC effluents has been developed by Rogers, Carreira, and co-workers (E2). The optical problems encountered in the design of flow cells for HPLC detection have been discussed by Stewart (F3). Sepaniak and Yeung have described a fiber-optic flow cell designed for HPLC detection by laser-induced fluorescence (F4). An apparatus for performing laser fluorescence, photoacoustic, and or photoionization detection in HPLC, using a windowless ow cell, has been described by Winefordner and co-workers (F5). A fluorescence detector for HPLC which uses a laser source and optical fiber waveguide effluent illumination arrangement has been described (F6). Detection in HPLC via fluorescence induced by electrons (emitted by a 147Pmpoint source) has been discussed; apparatus for such a detection scheme has been assembled and evaluated (F7, FB). Time-resolved fluorescence has been applied to enhance the selectivity of fluorometric HPLC detection for cases in which incomplete separation of fluorescent sample constituents is likely to prevail (F9). Another approach to selectivity in HPLC detection is to add a quencher to the eluent, choosing a substance which quenches the fluorescence of some analytes much more efficiently than others (FIO). The perennial problem of using fluorescence to detect nonfluorescent compounds eluting from HPLC columns has been attacked in a variety of ways. An extensive compendium of analytical derivatization reactions includes many fluorescence procedures for use in HPLC (F11). A general review on fluorescence derivatization procedures by Seitz considers fluorometric HPLC detection in considerable detail (B1). Lloyd has noted that many fluorescence derivatization procedures described in the literature cannot be assessed fully because the spectroscopiccharacteristics of the derivative are not set forth in sufficient detail and suggests the quantity E @ ~ / Aas w a “figure of merit” for fluorescent derivatives, wherein E is molar absorptivity at the excitation wavelength and Aw is the width of the fluorescence band (F12). Winefordner and co-workers have described a novel HPLC detection scheme wherein the mobile phase is spiked with a fluorophore such as aniline. Nonfluorescent sample constituents attenuate the fluorescence of this additive by quenching and/or inner-filter effects, whereas fluorescent analytes increase the fluorescence (either by their own fluorescenceor sensitization of additive fluorescence). Thus, unlike most fluorescence detectors, a response is obtained for nonfluorescent compounds without chemical derivatization (F13). The possibility that the additive may degrade the chromatographic resolution must, of course, be considered. Production of fluorescent products by postcolumn photolysis of nonfluorescent eluents has been used in HPLC detection; the advantages of this procedure over more conventional postcolumn derivatizations include reduced postcolumn chromatographic band broadening (F14-F16).A postcolumn system which enables the pH of the eluent to be changed rapidly can be used to maximize the fluorescence response for acidic or basic analytes, provided that their retention times and the pH dependences of their fluorescence spectra and quantum yields are known (F17). A novel scheme for fluorescent derivatization of metal ions entails use of an aminophenyl derivative of EDTA as a precolumn complexing agent; a postcolumn derivatization reaction of the uncomplexed amino group on the complexed ligand with fluorescamine generates the fluorescence (F18).A fluorescence detection procedure for organosulfur compounds proceeds by a POstcolumn ligand-exchangereaction of an eluted analyte with the Pd(I1)-calcein complex; the complexed ligand is nonfluorescent but the free ligand is intensely fluorescent, so release of the ligand generates a fluorescence signal. Such a procedure would be applicable in principle to any nonfluorescent polar compound which is a reasonably strong complexing ligand (F19).Frei and co-workers have described a modification of their ion-pair extraction derivatization technique wherein the ion-pairing reagent is added precolumn,

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rather than postcolumn as in previous designs. The extraction of the fluorescent ion pair into a solvent in which neither the analyte nor counterion are soluble proceeds, as in previous designs, postcolumn (F20). A comparison of the effects of air vs. solvent segmentation in postcolumn ion-pair extraction derivatization systems has been reported (F21). Solvent segmentation has been considered in conjunction with fluorometric HPLC detection schemes employing relatively slow postcolumn derivatization reactions (F22). Chemiluminescence(CL) is beginning to attract significant attention as a detection method in HPLC. A CL detection device for HPLC, in which the column eluent is nebulized by a high-velocity mixture of O3and Oz, has been described. Ozone-induced CL occurs with high efficiency only for certain classes of organic compounds; hence, this detector is not universal (F2.3).Another CL detector for HPLC uses a “serpentine” flow cell for CL generation; it has been applied to determination of ascorbic acid via the lucigenin CL system (F24). CL reactions of dansylamino acids (F25) or fluorescein-labeled catecholamines (F26) with bis(2,4,6-trichloropheny1)oxalate and H 2 0 are used for detection of the amino acids or amines in HPLk column effluents. Apparatus and procedures for postcolumn addition of reagents for bioluminescence detection (luciferin-luciferase)in HPLC have been described (F2‘f). Derivatization of microparticulate silica gel with dansyl chloride produces a fluorescent chemically modified surface. Spectroscopic studierg of such materials may be useful in elucidating the behavior of surface-bound entities and the nature of interactions of solvents with nonpolar bonded stationary phases emplalyed in HPLC (F28). The fundamentals of quantitative detection in thin-layer chromatography (TLC) by absorption, fluorescence, and fluorescence quenching have been reviewed; the importance of using high-performance plates and proper sample-application techniques is stressed (F29). Use of a SIT vidicon as a multichannel detector for acquiring spatially resolved emission spectra of spots on TLC plates has been reported (E5). Use of an Ar+ lager to visualize fluorescent spots on TLC plates reportedly produces greater sensitivity than conventional UV-lamp illumination (F30). A comparison of several commercial instruments for quantitative measurements of fluorescent compounlds on TLC plates, and a detailed discussion of spray and reagents and ancillary techniques for this purpose, has appeared (F31). The ideal characteristics of fluorescent spray reagents for TLC detection have been enumerated (F32). Mathematical models for quantitation of fluorescence from TLC plates continue to receive attention (F33). Fluorescence and chemiluminescence detection in gas chromatography (GC) has received some attention. Apparatus for reacting o-phthalnldehyde with primary amines eluting from a GC column has been designed (F34). A sulfur-selective GC detector, wherein S-containing compounds eluting from the column are converted to Sz in an oven flow cell, has been described. The fluorescence of S2is measured (F35). A CL system for GC detection is based on the reaction of hydrocarbons with “active nitrogen” (produced in a microwave cavity) to generate emission from excited CN molecules (F36). Nitrosamines eluting from a GC column are detected by pyrolysis to NO, followled by the CL reaction of NO with O3 (F37).

LOW-TEMPERATURE LUMINESCENCE Wehry and Mamantov have reviewed the principles, methodology, and analytical applications of frozen-solution and matrix-isolation cryogenic fluorometry ( G I ) . Techniques. Use of organic solvents, rather than inert gases, in matrix isolation fluorometry can often provide greatly enhanced selectivity for characterization of mixtures, especially if laser excitation is employed (C62, G2). The general advantages of using laser (rather than lamp) excitation in matrix-isolation fluorometry have been summarized (C65). The selectivity of matrix-islolation fluorometry of mixtures can be enhanced further by photoselection, in which a polarizer is used to selectively transmit the fluorescence of one component in a mixture of fluoro hores excited with polarized light (G3). Yang, D’Silva, anfFasse1 have demonstrated that use of dye-laser excitation in Shpol’skii frozen-solutionfluorometry, coupled with the use olf temperatures of ca. 15 K (rather than

the more common 77 K) effects very dramatic improvements in analytical selectivity (C63, C67, C69). The use of a deuterated analogue of a polycyclic aromatic hydrocarbon (PAH) as an internal standard in the laser-induced Shpol’skii fluorometric determination of that PAH has been demonstrated; the isotopic substitution produces a sufficiently large spectral shift to eliminate overlap of the quasilinear fluorescence and excitation spectra of the analyte and deuterated standard C69). Aromatic molecules, such as flavins, which ordinarily would not be expected to exhibit the Shpol’skii effect, may exhibit quasi-linear spectra in frozen n-alkane solutions if they are first derivatizecl with an alkyl group comparable in length to that of the matrix molecules (G4). A detailed quantitative study of Shpol’skii frozen-solution lluorometry indicates that solute aggregation and site distribution irreproducibility can be suppressed by using reproducible freezinglrocedures and ensuring that only very dilute solutions (10 M or less) are frozen (C67). The analytical selectivity of lamp-excited Shpol’skii fluorometry continues to receive scrutiny (G5). Small and co-workers have reported applications of “fluorescence line narrowing” (also known as “optical site selection” and “transition-energy selection” by other invesiigators) in aqueous glycerol glassy frozen solutions to the observation of highly resolved fluorescence spectra of individual polycyclic aromatic hydrocarbons in mixtures (034, (336). Personov and co-workers have discussed the principles of such experiments and have demonstrated the detection of individual PAHs in complex samples (e.g., gasoline) without prior separation. Quantitative aspects of the measurements also have been discussed (C68). Maple and Wehry have employed such an optical site-selection technique for identification and determination of hydroxyl-derivativesof PAHs in vapor-deposited fluorocarbon and inert gas matrices (G2). Application of the comparative method for determination obviously requires the of fluorescence quantum yield (aPF) txistence of a standard of known aF.Gibson and Rest contend that aFfor 9,10-diphenylanthraceneis -1.0 in a wide variety of solid matrices a t 12 K (G6). This observation, if correct, mggests that diphenylanthracene might serve as a quantumyield standard in low-temperature media, and the dearth of (€JFdata for fluorophores in such matrices might be alleviated without recourse to absolute @F determinations. The use of excitation-emission matrices in low-temperature phosphorescence spectrometry has been discussed (EI6). Conduction and immersion cooling devices for phosphoroinetry at 77 K have been compared (G7, G8). A sample compartment and an internal standardization technique for performing quantitative fluorescence measurements on inembranes or Dhotosvnthetic cells at 77 K have been described (G9). Applications. X-ray excited luminescence in Shpol’skii frozen solutions has been amlied to coal conversion and shale oil samples (G10). Shpol’&i fluorometry at 4.2 K following several chromatographic fractionations has been used to identify polycyclic aromatic hydrocarbons in medicinal “white oils” (GI1) and petroleum fractions and marine sediments (G12). Eastwood has reviewed the use of low-temperature luminescence in oil identification (EIO).The use of conventional and excitation-emission matrix phosphorescence methods in oil identification has been discussed (E16). Recent applications of phosphorescence to organic analysis have been reviewed by Winefordner and co-workers (G13). Among reported analytical applications of phosphorescence at 77 K are determinations of 6-mercaptopurine in blood plasma (GI4),2-amino-5-nitrobenzophenonein a tranquilizer preparation (GI@,@-glucuronidasein serum, urine, or tissue (GI@, and dopamine P-hydroxylase in rat plasma (G17).

ROOM-TEMPERATURE PHOSPHORESCENCE AND SOLID-SURFACE LUMINESCENCE Miller has provided a brief overview of room-temperature phosphorescence (RTP) on solid surfaces and in micellar solutions ( H I ) . Cline Love and co-workers have exploited the fact that the efficiency of phosphorescence in fluid solution often is enhanced when the analyte is incorporated into a micelle. Apparatus, techniques, and detection limits for RTP of several aromatic hydrocarbons solubilized in lauryl sulfate micelles in the presence of heavy atoms have been reported (H2-H4). Provided that they arise from scattering and/or ANALYTICAL CHEMISTRY, VOL. 54, NO. 5, APRIL 1982

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fluorescence, background signals in micellar RTP can be corrected for by win O2as a triplet quencher and subtracting the “blank” observef for an oxygenated sample solution from the signal observed for the same solution after deaeration. This approach fails, however, if the major source of background is impurity RTP or fluorescence which is quenched efficiently by O2(H5). Possible sources of error in the measurement and calculation of decay times for micellar RTP have been considered (H6).The possibility that RTP can be measured in deoxygenated liquid solutions in the absence of surfactants has been discussed; in general, however, the quantum yields are too small for realistic analytical application, although use of sensitized phosphorescence may offer some hope (H7). RTP of compounds adsorbed on solid surfaces has been The common tendency for solid-surface reviewed (H8-HIO). RTP signals to be attenuated by O2and H 2 0 can be diminished by treatin the support, or samples spotted thereon, with sodium citrate fH11). The background phosphorescence observed from virtually all filter papers is a problem for which no solution appears yet to have emerged; washing or heating the paper prior to use does not seem to suffice (H12). Application of solid-surface fluorescence methodology to homogeneous substrate-labeled fluoroimmunoassays has been reported (H13). Reactions with 2-diphenylacetyl-1,3indandione-l-imine derivatives on solid surfaces, to form fluorescent products, can be employed for determination of various common pesticides (H14). A spot-test procedure on filter paper, employing sensitized fluorescence, is used for detection of polycyclic aromatic hydrocarbons in field studies (H15). RTP on filter-paper substrates has been applied to H16) determination of polycyclic aromatics in coal liquids (E9, and airborne particulate samples (E8). Pflug and Faulkner have designed some very pretty fluorometric techniques for use in studying species adsorbed on electrodes and undergoing electrochemical reactions (HI 7). The surface-enhanced Raman phenomenon has, not surprisingly, stimulated studies on the fluorescence of organic (H18)and inorganic (H19)species adsorbed on metallic surfaces. Spectral shifts, energy transfer, and enhanced emission intensities (under certain conditions) are observed. Phosphorescence quantum yields for organic molecules adsorbed on metallic surfaces are measured as a function of the distance of the molecule from the metal by using a layer of condensed argon as a “variable spacer” (H20).

POLARIZED FLUORESCENCE AND RELATED PHENOMENA Instrumentation and Techniques. A fluorescence polarization spectrometer employing a continuously rotating polarizer, a single photomultiplier, and lock-in detection has been described, and its advantages over dual-detector instruments have been discussed (11). Modification of a conventional fluorometer for polarization measurements has been described; a novel component is a “polarization chopper” containing two horizontally and two vertically oriented polarizers in a rotating can assembly (12). An epi-illumination arrangement for measurements of fluorescence polarization in flow systems has been described (13). Detailed descriptions of automated instrumentation for use in fluorescence polarization immunoarrays have appeared (14,15). Techniques for performing accurate measurements of fluorescence polarization of samples under high pressure have been discussed (16). A polarization rotator constructed from twisted nematic liquid crystals has been described (17). Instrumentation for measurement of time-resolved fluorescence anisotropies in the picosecond regime has been described (18). A number of reports have discussed “fluorescence-detected circular dichroism”, wherein the difference in fluorescence intensity produced by excitation with left and right circularly polarized light is measured. Lobenstine and Turner have argued (in opposition to some previous reports) that fluorescence-detected CD spectra can be compared directly with conventional CD spectra, provided that artifacts due to linear polarization are eliminated by suitable instrument design and data treatment (19). Instrumentation and techniques for the performance of fluorescence detected circular dichroism have been described (110). Instrumentation and techniques for “fluorescence-detectedlinear dichroism” have been described, and applications of the technique to study 138R


molecular orientations in anisotropic systems have been discussed (111). Applications. A review of the use of polarized light spectroscopy (including steady-state and time-resolved fluorescence polarization measurements) to study the orientation and mobility of molecules in membranes has appeared (112). The use of polarized fluorescence measurements for the determination of individual compounds in binary mixtures whose excitation and/or emission spectra overlap continues to receive discussion and demonstration (113). The use of photoselection techniques to discriminate between fluorophores whose low-temperature fluorescence spectra overlap has been discussed by Maple and Wehry (G3). Fluorescence polarization assays of enzymes using substrates labeled with fluorescein isothiocyanate have been described; the fluorescence polarization of the substrate decreases as the substrate is converted to products (114).

KINETIC FLUOROMETRIC METHODS OF ANALYSIS The principles and practice of kinetic fluorometric methods of analysis have been reviewed exhaustively by Ingle and Ryan (C142). Modifications to a commercial stopped-flow apparatus for fluorescence detection have been described (J1).Kinetic fluorometric assays for several proteinase enzymes have been carried out by using a fluorometric fast centrifugal analyzer (C151). A kinetic enzymatic method for serum triglycerides has been described (J2). Arylsulfatase enzymes are determined by a rate method based on their catalysis of hydrolysis of 2-naphthyl sulfate to 2-naphthol. The naphthol undergoes excited-state dissociation, and the fluorescence spectrum of the naphtholate is well to the red of that of the unreacted substrate (J3). The use of kinetic fluorometricmeasurements in flow cytometry has been discussed (54). The oxidation by Hg2+ of nonfluorescent thiamine to a fluorescent product is the basis of a kinetic fluorometric technique for thiamine determination (J5). Fixed-time, initial-rate, and fixed-intensitykinetic methods for Ti(IV), based on its catalysis of the oxidation of picolinaldehyde nicotinoylhydrazone to form a fluorescent product, have been reported (J6). A kinetic method for V(V) in petroleum crudes is based on its reaction with l-amino-4-hydroxyanthraquinone, to produce fluorescent oxidation products (J7). Another kinetic method for V(V) is based upon vanadate catalysis of autoxidation of 4,8-diamino-1,5-dihydroxyanthraquinone2,6-disulfonate (J8).The rate of complex formation with 2,2’-bipyridine ketone azine is employed in a fluorometric determination of Au (J9). The copper(I1) catalyzed autoxidation of 2,2’-bipyridine ketone hydrazone, which produces fluorescent products, has been employed in a kinetic determination of Cu(I1) (JIO). Fe(II1) and Tl(II1) react with 1,4diamin0-2~3-dihydroxyanthraquinone to generate a fluorescent product, and this reaction is the basis of a kinetic method reported for the determination of these cations ( J I I ) . A “response surface” optimization study of a fluorometric enzymatic method for As(1V) determinations is illustrative of the utility of “chemometrics” in kinetic fluorometric methods (512). TIME-RESOLVED LUMINESCENCE SPECTROMETRY The literature pertaining to experimentaltechniques in fast luminescence spectrometry is truly Brobdingnagian;although analytical applications of time-resolved luminescence are being reported at a much slower pace, a moderately thorough coverage of analytically relevant recent developments in experimental technique is attempted in this section. Instrumentation and Experimental Techniques. Several useful reviews of experimental techniques in very fast fluorescence spectroscopy have appeared ( K 1 4 3 ) . Hieftje and Vogelstein have provided a useful overview of the various measurement approaches in time-resolved fluorometry, with special emphasis on correlation-based methods and the applications of linear response theory to fast fluorescence spectroscopy (K4). Hieftje and Haugen have discussed in considerable detail the use of correlation techniques in time-resolved fluorometry. Several different measurement schemes have been delineated; in general, their use enables accurate decay-time data to be obtained via the use of simpler instrumentation than that


required for the conventional pulsed-excitationmethods (K5). A double-beam modulation time-resolved fluorometer has been described, and its use to measure small changes in fluorescence decay times (produced, for example, by quenching) is discussed (K6). A mathematical anailysis of possible sources of error in a modulation procedure for fluorescence decay-time measurements using a CW laser has been presented; it is concluded that the technique should be capable of decay time measurement in the picosecond domain (K7). Weber has published a mathematical formulation for extracting the decay times and relative intensities of a mixture of N fluorophores via phase fluorometry using N different frequencies of modulation of the incident light (K8). An experimental test of this approach to a twocomponent system (tryptophan zwitterion and anion) has been reported (K9). These two papers discuss in some detail ithe relative merits of pulse and phase fluorometry, at the present state of the art,for samples which do not exhibit clean single-exponential decays. Correction for “color delay” errors in phase fluorometry also is considered. Equations have been derived relating the “fluorescence saturation factor” to the fluorescence decay time of a fluorophore. Under certain conditions, measurement of the fluorescence saturation factor as a function of incident photon flux (using spontaneous Raman scattering of the solvent as an internal standard) pirovides data from which the decay time can be calculated. Applications of the technique to deterl ~conmination of TF for rhodamine laser dyes at ~ 1 0 - M centrations are reported. The method requires a sufficiently powerful laser to produce saturation (but stimulated Raman scattering from the solvent must be avoided). The method is potentially very attractive because a short-pulse laser is not required, nor are sophisticated fast signal detection systems (KlO). A “pump and probe” pulse technique has been described for the measurement of subnanosecond fluorescence decay curves. One “pump” and two “probe” pulses of the same wavelength (from a synchronously pumped dye. laser) are caused to impinge upon the sample, with the time delay between the pump pulse and one of the probe pulses being varied. The manner in which the difference signal depends upon the time delay between the pulses is used to extract the fluorescence decay curve for the sample (K11). Under certain circumstances in pulse fluorometry, the fluorescence decay tirne for a species can be obtained by measuring the time interval between the maximum in the excitation light pulse and the maximum in the resulting fluorescence pulse. The assumptions in this approach, and techniques for implementing it, are discussed by O’Dowd et al. (K12). Phillips and co-worhers have discussed pulse methods for fluorescence decay-time measurements by time-correlated photon counting. Thei3e authors have concluded that a cavity-dumped CW laser may in many cases be superior for this purpose to a mode-locked laser, despite the much greater pulse durations characteristic of the former, because the cavitydumped laser is less expensive, easier to use, and produces pulse profiles of greater stability than a mode-locked laser (K13). In pulse fluorometry, the widths and shapes of the pulses produced by the source are of course crucial; developmentand characterization of pulsed laser systems accordingly continue to attract widespread attention. Harris has reviewed the fundamentals arid characteristics of pulsed laser systems (C7). Mode-locked lasers have a reputation for being touchy, unreliable, and difficult to operate; an acousto-optic mode-locked laser system which is reported to require minimal operator attention after the initial setup process has been described (K14). A system for electro-optic pulse picking from the ulse train of a mode-locked Ar+ laser has been described in fetail (K15). A N2-pumped dye laser reportedly capable of generating 2 ps pulses uses ia modified Michelson interferometer as active mode locker and wavelength tuning device (K16). Synchronouslypumpled dye lasers are receiving widespread attention for use in time-resolved fluorometry. The shape and intensity of pulses from a synchronously pumped dye laser have been studied as a function of cavity parameters and the length of the pump laser pulses; techniques for improving the quality of the dye laser pulses are discussed (K17). A streak-camerastudy of individual pulses from a synchronously

pumped dye laser a3 a function of the dye laser cavity length reveals the existence of satellite pulses which are difficult to detect by other detection procedures. It is recommended that the dye laser be operated at a cavity length several micrometers greater than the matched position to minimize these difficulties (K18). Techniques for decreasing pulse-to-pulse width (K19)and amplitude (K20)variations in synchronously pumped dye lasers have been described. The long-term stability of output of a synchronously pumped dye laser can be enhanced by active stabilization of the optical length of the lalser cavity; a technique for accomplishing this has been described (K21). A synchronously pumped cavity-dumped mode-locked dye laser using a ring cavity configuration is dlescribed, and the advantages of the ring cavity for synclironous mode-loclrin are described (K22). Generation of subpicosecond pulses y synchronous pumping of ring dye lasers with a mode-locked Ar+ laser has been reported (K23, K24). Production of picosecond pulses from a cavity-dumped synchronously pumped dye laser fitted with intracavity frequency-doubling crystals has been discussed (K26).The pulse characteristics of a mode-locked Kr+ laser at six wavelengths (ranging from 520.8 to 752.5 nm) have been discussed, and use of this laser to synchronously pump a dye laser has been advocated (K27). Use of the second harmonic of a modelocked NdYAG laser to synchronously pump a dye laser has bleen reported, and possible advantages of this scheme over the more common use of Ar+ as pump have been outlined (K28). A scheme termed “reciprocal synchronous passive mode locking” can be employed to produce picosecond pulses from a synchronously pumped dye laser; in this procedure, the Ar+ pump laser is passively mode-locked by the saturable absorption and lasing action of the dye, so it is not necessary to use an acousto-optically mode-locked pump laser (K29). The use of a XeCl excimer laser to amplify the frequency-doubled output of a mode-locked dye laser has been reported (K30). Production of subpicosecondpulses at 308 nm b,y active mode locking of a XeCl excimer laser has been reported (K31). Use of a Michelson interferometer for active mode-locking of an argon-pumped dye laser has been discussed (K32). Production of ca. 3 ps pulses from a mode-locked transverse-flow dye laser using flash lamps as the pump source has been discussed (K33). A transverse gas flow atmospheric-pressure N2 laser is reported to produce pulses of ca. 0.75 ns pulse width at repetition rates approaching 1kHz; use of this laser for dye-laser pumping and time-resolved fluorometry has been discussed (K34,K35). Pumping of a dye laser with an atmospheric-pressure N2 laser (0.5 ns pulse width) can produce 100-ps pulses from the dye laser, apparently due to amplified stimulated emission phenomena in the dye (K36). Use of a reflecting Fizeau wedge as a tuning element in a dye laser may (in comparison with etalons) have advantages, particularly when short pump pulses are used (K37). Munro and Sabersky have reviewed the characteristics of synchrotron radiation and its application to measurement of fluorescence decay times and time-resolved fluorescence spectra K38). In very fast spectroscopy,one encounters the problem that monochromators act as pulse broadeners due to optical path dnfferences in the monochromator; this effect has been measured and is reported to be potentially serious for high-resoluition grating monochromators (K39). The use of a grid-gated PMT to perform time-resolved fluorescence measurements in the ca. 0.5 ns time regime has been demonstrated (K40). Gustafson and Lytle have described a scheme involving use of a “double-balancedmixer” atr a fast gate for gated photon counting at repetition rates in the MHz regime, The advantage of this system is that, unlike most other photon-counting schemes used in time-resolved fluorometry, advantage is taken of the high pulse repetition rates available from ion lasers ( K 4 1 ) . A microcomputer-controlled instrument for decay curve measurements via time-correlatedphoton counting has been described (K42). Degradation in time resolution and changes in calibration of time-to-amplitude converters may occur at high conversion rates; these effects may be significant in fluorescence lifetime measurements employing the time-correlatedphoton-counting technique (K43). Various procedures for gating TV camera tube electronic array detector systems to be used for timeresolved spectroscopy in the nanosecond to picosecond regime have been described (C137,K44). The problem of capacitive

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lag in SIT vidicons, and its effects upon the measurement of fast optical signals using these detectors, have been discussed (K45). Modern high-speed detectors may produce photocurrent pulses which are temporally so short as to render “conventional” electronic signal processing and measurement procedures inapplicable; several novel approaches for dealing with the electronic aspects of ultrafast optical signals have been described (K46). The noise in fluorescence decay measurements employing sampling oscilloscopes is reported to obey Poisson statistics (K47). A detailed discussion of hardware and software of interfacing a sampling oscilloscope to a minicomputer has appeared (K48).Studies of the dynamic range of various streak camera systems have appeared (K49). Detection limits and linear dynamic ranges for several common fluorophores obtained with a pulsed nitrogen laser and streak camera detector have been reported (K50). Use of a vidicon tube (rather than photographic film) as the recording medium is reported to effect an increase in the dynamic range of a picosecond streak camera (K51, K52). Techniques for measurement of time-resolved fluorescence anisotropies in the picosecond regime have been described (18). An instrument designed to measure luminescence lifetimes in the microsecond to second range has been described, and its use for measurement of phosphorescence lifetimes in room-temperature solutions containing micellar triplet stabilizers has been discussed (H4). Deconvolution and Related Matters. The problems of obtaining “true” fluorescence decay times and time-resolved spectra when the source decay is not very short relative to the luminescence decay are, as always, a focus of concern. Phillips and co-workers have compared time-resolved fluorescence spectra of several two-component mixtures, measured via time-correlated photon counting with and without deconvolution of the source emission profile. They have concluded that deconvolution of the decays followed by reconstruction of the time-resolved spectrum almost inevitably effects a loss of spectral resolution, but failure to deconvolute generates incorrect kinetic information (though generally producing spectra of higher quality) (K53). Given that the “deconvolute and reconstruct” procedure is very slow, there is probably no reason to employ it unless accurate decay kinetics are required (which, for most analytical applications of time-resolved fluorometry, would not be the case). Because the source decay function often is wavelengthdependent, there are advantages in avoiding its measurement. Ricka has defined a procedure called “relative deconvolution”, which requires two or more measurements of the fluorescence decay function a t the same excitation and emission wavelength. In measurements of time-resolved fluorescence anisotropies or fluorescence quenching, this condition is easy to satisfy. The mathematical arguments underlying this approach have been set forth, and some example data have been analyzed. This method may be very tricky to use in cases in which a single exponential fluorescence decay is not observed (K54). A method of moments procedure for analyzing fluorescence decay curves having the functional form exp(-At - Btliz) is described. In conjunction with “moment index displacement”, this method is said to achieve correction of decay-time data for Rayleigh scattering, lamp intensit drift, and zero-point displacements (K55). Cline Love and 8 a v e r have concluded that reiterative deconvolution procedures can provide decay times for single exponential decays with standard deviations as small as 0.02 ns under certain conditions; resolution of two-component exponential decays by this procedure requires a minimum lifetime difference for the two components of 3 ns (K56). The ”phase plane” method for deconvoluticm of decay time data has been described, and its accuracy for deconvolution of single exponential decays has been assessed (K57). A nomogram for deconvolving single-exponential fluorescence decays from the decay function for typical commercial N2flashlamps has been presented; such an approach, while not highly accurate, is very rapid (K58). Criteria for “goodness of fit” in deconvolution computations have been set forth (K59). The use of factor analysis techniques in the interpretation of fluorescence decay time data for series of homologous compounds has been discussed (K60). Analytical Applications. Hieftje and Haugen have discussed the elimination of quenching as a major source of error in analytical fluorescence spectrometry via time resolution. 140R


The technique involves simultaneous (or consecutive) measurement of the fluorescence intensity and decay time, each of which should be altered by the same factor if quenching occurs; hence, the ratio of intensity to decay time should be unaffected by quenching. An instrument for acquiring all necessary data to implement this approach also is described (K61). Knorr and Harris have discussed the use of time-resolved fluorescence measurements for resolution of overlapping fluorescence spectra in multicomponent samples. This approach involves generation of a data matrix via measurement of fluorescence spectra of a sample as a function of time following excitation with a pulsed laser. The matrix is then decomposed into a “spectral” matrix and a “decay time” matrix, which can be used to identify sample constituents according to their characteristic spectral and temporal properties. The procedure, which is formally equivalent to performing a reiterative convolution of the fluorescence decay curve at each wavelength in the spectral scan, is reported to be useful for resolving spectrally overlapped fluorescence spectra of multicomponent samples without requiring advance knowledge of the identities of the sample constituents (K62). The detection limits of most fluorometric analyses are established by the blank (impurity luminescence, scattering, luminescence from instrument components, etc.), and it has long been recognized that time-resolved detection of fluorescence offers potential improvement in detection limits as well as enhanced selectivity. Haugen and Lytle have provided an interesting discussion of how this might be achieved, via use of the concept of “time filtering”. They also have considered some of the practical problems encountered in implementing temporal discrimination against blanks in fluorometric analyses (K63). When a synchronously pumped dye laser was used as excitation source, to provide temporal discrimination against scattered source light (and perhaps some background luminescence), the detection limit for fluorometric A13+ determination, via the lumogallion complexation procedure, was found to be 60 ng/L. To achieve such a low detection limit, it was necessary to use ultrapure water as solvent, and whether these results will carry over to real samples remains to be ascertained (K64). Use of timeresolved fluorometry to distinguish signals from scattered light in laser-induced fluorescence analysis (C74)and fluorescence photobleaching recovery experiments (K40) has been described. Cline Love and Upton have considered the analysis of binary mixtures of compounds whose fluorescence spectra overlap by fluorescence decay-time measurements using reiterative convolution computations; the conditions which must be satisfied in order for reliable quantitative results to be obtained are delineated (K65). The possibility that the fluorescence decay times of structurally similar complex organic molecules, differing only in the nature or positions of substituent groups, may be sufficiently varied to enable them to be used as diagnostic criteria for identification of the compounds in question, has been considered (K66). Richardson and co-workers have used time resolution to enhance the selectivity of fluorometric detection in the liquid chromatography of mixtures of polycyclic aromatic hydrocarbons (F9).Time resolution has been employed in the luminescence determination of UOZ2+in aquatic samples to eliminate interference from fluorescent organic constituents of the samples, based upon the long decay time of UOZ2+luminescence (microsecond)compared with the nanosecond duration decays of the fluorescent organic interferents (K67, K68).

CHEMILUMINESCENCE Books and Reviews. DeLuca and McElros have edited the proceedings of the second International Symposium on Bioluminescenceand Chemiluminescence,consisting of papers dealing both with basic chemibtry and analytical applications of chemiluminescence (CL) and bioluminescence (BL) phenomena (L1). Schram and Stanley have edited the proceedings of a symposium on analytical applications of BL and CL (L.2). Apparatus and Experimental Techniques. Microcomputer-controlled reagent dilution systems (L3, L4) and a microcomputer-controlled CL photometer (L5) have been described. A microprocessor control and data aquisition for a CL photometer has been described in detail (L6). The


instrumental problems associated with the detection of weak CL signals, and the use of photon-countingtechniques in such situations, have been discussed in detail by Seliger (C128). Measurement of CL iepectra is difficult with “conventional” instrumentation because of the transient nature of many CL signals. Marino and Ingle have described a microprocessorcontrolled diode-array spectrograph capable of producing spectra of chemiluminescent systems wherein the lifetime of the CL signal is ca. 1 s or less (C143). Spatially resolved CL or BL measurements represent one possible application of an image intensifier-vidicon microscope described by Rich and Wampler (E7). A portable centrifugal fast analyzer suitable for CL kinetic analyses has been described (C152). The use of flow-injection techniques in solution CL has received considerableattention (L7,Ls).A CL flow photometer with provision for coulometric reagent generation haf, been described (L9). Seitz has presented a theoretical analysis of optimal flow-cell designs for solution CL measurements (L10). Wampler and co-workers have noted that solutiion-phase CL may be quite spatially anisotropic, and this fact should be considered in the design of cells and measurement systems for CL (L11). Microporous membrane CL flow cells have been described (L12),and the spatial profile of light emission from such cells has been analyzed (L13). Instrumentation for performance of titrations in which the end point is detected by CL has been described (L14). A battery-operated CL photometer, suitable for “in the field” use, has been designed (L15). A portable CL photometer for use in H202determinations in natural waters via the lumin ~ l - F e ( c N ) ~reaction ~has been described (L16). The recipe for a quenched liquid scintillator solution which contains a tritiated organic compound, and reportedly produces one photon per radioactive decay event, has been given, and its use to standardize CL and BL measurements has been reported (L17). The limitations imposed on BL analyses by impurities in the various enzymes have long been recognized as severe. If sufficientlly pure luciferase is used, BL analyses for ADP and AMP now are not limited by the luciferase itself but by impurities in the enzymes required for conversion of ADP and AMP to ATP (L18). A comparison of commercial firefly luciferase preparations has been described (L19). Covalent attachment of bioluminescent enzymes (including luciferase) to “Sepharose”is reported to produce much greater enzyme activitv than immobilization on chemically modified glass beads (LZO). The use of simplex optimizations in the development of solution CL txocedures has been discussed and shown to be useful in ascertaining optimum reagent concentrations much more rapidly than is possible by manual techniques (L5). The utility of time-resolved spectrometry in the stud of bioluminescence phenomena has been discussed (L21 Analytical Applications of Gas-Phase CL. The reliability of gas-phase CL procedures continues to be a matter of concern. It has been concluded that CL determination of nitrogen oxides (“NO,”) is more selective and precise than other methods for determining these materials, provided that periodic recalibration of the instrumentation is carried out (L22). Commercial CL analyzers are repwted to produce results which are too low, when used for NO determinations in stack gases (L23). In the gas-phase CL determination of nitrosamines (via cleavage therefrom of NO followed by CL interference by amines, resulting reaction of the latter with 03), in suppression of the CIL response, has beqn noted; chemical destruction of potential amine interferents prior to CL determination of nitrosamines is recommended (L24). The O3 CL reaction with ethylene produces formaldehyde in stoichiometric quantities, which can be det,ermined by absorptiometry. Such data can be used to calibrate the O3response of this type of CL O3 imeter (L25). The chemiluminescent reaction of NO and NO2 with hydrogen atoms (produced by thermal cracking of H,) is the basis points of superiority of this apof a CL monitor for “NO,”; proach over other CL monitoring systems for NO and NO2 have been advanced (126). The determination of traces of NO in aqueous solutions by stripping from solution into the gas phase followed by CL measurement, using O3 as the reagent gas, has been described (L27). A CL procedure for NO3- and NO2- proceeds via their reduction to NO, which is then determined via thle O3 reaction (L28). Application of a

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commercial “NO,” CL analyzer to determination of oxides of nitrogen in cigarette smoke has been reported (L29). Cured meats containing nitrite can be treated with acid, coverting the NOT to NO, which can be determined by gas-phase CL; comparisons of the results so obtained with these from differential pulse polarographic and absorptiometric determinations are presented (L30). Total bound nitrogen in organic compounds and biological samples reportedly can be determined by pyrolysis of the sample to produce NO, which is then determined via the CL reaction with O3 (L31,,532). Elemental analysis for nitrogen in petroleum can be performed by burning the sample and determining the NO so produced by CL (L33). The CL reaction of luminol (as an aerosol) with C12is the basis of a method for atmospheric chlorine determinations (L34). The CL measurement of part-per-billion levels of Ni(CO)., in air via reaction with O3 and CO has been described (L35). The CL reactions of O3with rhodamine B (1536) or 2-methyl-2-butene (L37) have been used in rocket- and ballalon-borne sensors for stratospheric O3 determinations. A mlonitor for “total reactive hydrocarbons” in air is based on the CL reaction with oxygen atoms (L38). Gas chromatography detectors based on various gas-phase CL reactions have been described (F36, F37). Analytical Applications of Solution CL. A CL determination of H 02!using paper impregnated with luminol as a solid-phase &L indicator, has been reported (1539). A collagen membrane containing immobilized peroxidase generates CL in the presence of luminol and H20z. This system can be used to detect HzOz,or (in principle) any substance which can be coupled to a reaction which generates Hz02 (L52). An O2 sensor based on the CL reaction of Oz with tetraaminoethylenes has been developed and its performance has been compared with that of Oz electrodes (L40). Another CL miethod for O2in aqueous solutions uses the reaction of alkaline luminol with Ozin the presence of Fe(CN) (L41). CL methods for determination of Clz (and/or Hob1 and OC1-) in water, based on the luminol system (L42) and on the H2OZ-OC1-“singlet Oz” reaction (L43) have been discussed and compared by Marino and Ingle. C102in aqueous solution has been determined by luminol CL (L12). A CL method for NOz, using the luminol reaction without a metal-ion catalyst, has been reported (L44). Determination of atmospheric SO2 via its apparent catalysis of oxidation of disulfitomercurate ion by Mn04- in acidic aqueous solutions has been reported (L45). CL determinations of sulfide in aqueous solution, via its CL reaction with coulometrically generated hypobromite (L9) OF peroxidase-catalyzed oxidation of S2-by H20zusing rhodamine B or fluorescein as sensitizers (L46),have been reported. The use of luminol as the indicator in the volumetric titration of As(II1) with OBr- has been described; CL (due to the presence of unreacted O B i ) which occurs at the end point signals the end of the titration (L47). The use of chelating ion-exchange resins for rapid removal of interfering ions has been advocated for selectivity improvement in solution CL determinations of metal ions ( L a ) . A flow-injection analysis scheme for determination of Zn(I1) and Cd(II), via their inhibition of Co(I1)-catalyzed luminol CL, has been described (L49). Determinations of Cr(II1) in seawater via luminol CL (L50) and Cr(V1) in natural waters b y lophine CL ( L 5 f )have been reported. Amines, such as NH3 and ethylenediamine, which complex with Ag+ can be determined via catalysis, by the complexes, of the luminol-SzO~-reaction (L53). Determination of humic acid in natural waters by its CL reaction with Mn0; has been discussed (L54). Determination of uric acid by peroxyoxalate CL has been described (L55). Several liquid-chromatography detectors based on chemiluminescence have been described (F23-F26). Stanley has reviewed clinical applications of CL and BL (L59). Campbell and Simpson have reviewed the use of CL and BL assay methods in biological systems (L60). A CL method for thiols is based upon an exchange reaction of a thiol (in a solution sample) with a thiol derivative of luminol bound by a dithiol linkage to a solid polysaccharide. The displaced luminol species is then determined by its CL reaction with H202 under conventional conditions (L56). An extension of this technique is employed to determine serum cholinesterase; in this case, the bound thiol derivative of luminol is displaced ANALYTICAL CHEMISTRY, VOL. 54, NO. 5, APRIL 1982

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from the solid phase by a thiol released by the enzyme (L57). Acetylcholine has been determined via its hydrolysis (by acetylcholinesterase)to choline, followed by oxidation of the the formation of choline (by choline oxidase) to form H 02, which is detected by luminol CL (L58p. A procedure for determination of hemoglobin and other hemoproteins in blood serum, via perborate-initiated luminol CL, has been described (L61).A novel enzyme assay technique involves the use of synthetic substrates which release isoluminol as a product of the enzyme-catalyzed reaction; the isoluminol is then determined via CL induced by H2020 3 2 ) . Inhibition of the superoxide-mediated CL of luminol by superoxide dismutase has been employed for determination of the enzyme in bovine ocular subfractions (L63). When arachidonic acid is injected into platelets containing luminol, CL results. The CL is inhibited by certain drugs. It is suggested that this response could be used to screen drugs for effectiveness and/or toxicity (L64). The use of CL procedures for monitoring the number of granulocytes present in whole human blood has been described, and the use of these data to detect defects in cellular defense mechanisms is discussed 6565). The use of CL in immunoassay has been reviewed (L66, L67). A sizable number of specific immunoassay procedures incorporating CL measurements has been reported; they are described in more detail in a later section. The use of CL and BL in competitive protein binding immunoassay procedures has been reviewed (L68). Luminol CL has been used to determine antibodies in serum samples (L69). Electrochemiluminescence. Although there is a large literature pertainin to mechanistic aspects of electrochemiluminescence (ECLf few analytical applications of ECL have ever been reported. The observation that the ratio of pulsed ECL intensities for successive positive and negative voltage pulses in the luminol system depehds on the Cu(I1) concentration of the solution is the basis of an ECL determination of Cu(I1) (L70). The possible application of ECL to the determination of individual polycyclic aromatic hydrocarbons in mixtures also has been discussed (L71). Whether such approaches have any significant analytical advantages over more conventional CL techniques is not yet clear. Analytical Applications of Bioluminescence. The chemistry (L72) and analytical applications (L73) of firefly BL have been summarized. An overview of BL applications (L59) is of interest. Some general aspects of potential applications of BL in cell biology and microanalysis have been discussed by Brolin and Wettermark (L74). A thorough review of energy-transfer processes in bioluminescence has been presented by Ward (L75). Oxygen determinations via a “membrane-covered photobacterium probe”,based upon the O2requirement for BL from Photobacterium fischeri, have been described;the membrane system is used to prevent contact of the luminescent bacteria with otential inhibitors (L76). Bioluminescence procedures baseJon the luciferin-ATP reaction have been used for determinations of myocardial high-energy phosphates (L77)and chloroamphenicol in serum (L78). An adaptation of the luciferin-luciferase BL system to the determination of serum triglycerides has been described (L79). BL methods for androsterone and testosterone, using luciferase and other enzymes co-immobilizedon “Sepharose 4 B , have been described (L20). A bioluminescence method for determining regional glucose distribution patterns in brain slices has been described; the BL reaction is based on the linkage of NADPH formation from glucose to a bioluminescent marine bacterium (L80).

FLUOROIMMUNOASSAY AND RELATED PROCEDURES Reviews. The use of fluorescence and chemiluminescence measurements in immunochemical assays continues to grow rapidly in both volume and sophistication. Two volumes of Methods i n Enzymology ( M l , M2) contain a number of articles dealing with basic principles and techniques in immunochemistry, including a detailed survey of the fundamentals of antigen-antibody reactions (M3). An introductory presentation of immunoassay principles has been presented by Chait and Ebersole (M4). A detailed review of the principles and practice of fluoroimmunoassay has been presented by Smith, Hassan, and Nargessi (M5). Landon and Kame1 have reviewed the use of fluorophore-tagged reagents in immu142R


noassay (M6). Smith has provided a brief, readable review of fluorescence polarization immunoassay (M7). Schall and Tenoso have reviewed “alternatives to radioimmunoassay”, including both fluorescence and chemiluminescence techniques (M8). The proceedings of a 1978 symposium on “immunoassays in the clinical laboratory” contain a number of chapters dealing with FIA procedures (M9). The second edition of a monograph on immunocytochemistry contains a detailed chapter dealing with immunofluorometric techniques (MIO). Enzyme immunoassays employing fluorescence and chemiluminescence procedures have been reviewed (M11). Boguslaski and co-workers have reviewed competitive protein binding assays, as monitored by chemiluminescence and bioluminescencetechniques (L68)and substrate-labeled homogeneous fluoroimmunoassays for haptens and proteins (M12). Ullman has reviewed the determination of serum proteins by homogeneous fluorescence and enzyme immunoassays (M13). Pecht has reviewed the use of intrinsic and extrinsic fluorescent probes to make inferences regarding the modes of antibody action (M14). Comparisons with Other Analytical Techniques. Considerable effort continues to be devoted to comparisons of the accuracy and/or precision of fluoroimmunoassay (FIA) with those of more established techniques, especially radioimmunoassay (RIA). The accuracy of serum phenytoin determination by substrate-labeled fluoroimmunoassay compares favorably with that obtained by several other analytical techniques, including RIA and liquid chromatography (M15). A comparison of the results of three methods (immunofluorescence, radial immunodiffusion, and nephelometry) for determination of serum immunoglobulinshas been reported (M16). The precision of one commercial fluorescence system and three commercial nephelometers for immunoglobulin quantitation in serum has been compared (M17). A comparison of the results of radioimmunoassay and a magnetizable solid-phase separation fluoroimmunoassayfor total estriol in serum has been reported (M18). A homogeneous substratelabeled FIA for theophylline in serum has been reported to produce results which correlate well with a gas chromatographic procedure and a commercial enzyme immunoassay for theophylline (M19).A modification of this procedure incorporates the reagents into a paper strip, and a solid-surface measurement format is used (H13). A heterogeneous fluorescence immunoassay for gentamicin in serum, employing a second-antibody separation, has been described, and the results of this assay have been compared with those of RIA (M20). A heterogeneous fluoroimmunoassay for progesterone in serum or plasma has been described, and a correlation of the results with those obtained via RIA is presented (M21). The results of a heterogeneous double-antibody FIA for tobramycin in serum samples have been compared with those obtained by RIA and a microbiological assay (M22). A polymeric solid-phase immunoadsorbent, used both as an immobilization substrate and an optical surface for fluorescence measurement, has been developed and used for assay of serum immunoglobulins. The procedure is compared with a commercial fluoroimmunoassay system, which requires in situ generation of the immunoadsorbent (M23). Fluorescent Labels and Energy-Transfer Immunoassays. The use of prolonged incubation times (ca. 20 h) enables generation of detectable fluorescence signals via use of decreased quantities of fluorescent conjugates. This result is important in terms of decreased cost for the more expensive fluorescent reagents. In addition, it is contended that the specificity of staining may be enhanced by use of a lengthy incubation, instead of the customary 1 h or less (M24). Fluorescein isothiocyanate (FITC) photodecomposesrapidly when exposed to 488-nm light from an argon laser. It appears that significant differences in the bleaching characteristics of FITC may be noted, depending upon whether or not the FITC conjugate is specifically bound. It is conceivable that such photobleaching effects may facilitate discrimination between specific and nonspecific staining (M25). Techniques for minimizing nonspecific staining of eosinophilic granulocytes in immunofluorescence studies employing fluorescein isothiocyanate or tetramethylrhodamine isothiocyanate as stains have been discussed (M26). An energy-transfer immunoassay for serum albumin has been developed, and the effects of instrumental and chemical parameters upon the detection limits and quantitative pre-

MOLECULAR FLUORESCENCE, PHOSPHORESCENCE. AND CHEMILUMINESCENCE SPECTROMETRY

cision of the method have been evaluated. It was concluded that new fluorescent labels must be developed before the energy-transfer immunoassay technique can be optimized (M27). The development of new donor-acceptor pairs for energy-transfer immunoassay indeed is attracting significant attention (M28). A modification of the energy-transfer fluoroimmunoassay procedure, employing fluorescamine as donor and fluorescein as acceptor, has been described (M29). Energy-transfer FIAs for thyroxine-binding globulin (M30) and proteins (M’31)have been described. Developments in Techniques and Applications of FIA. Zare and co-workers have provided detailed descriptions of a laser-induced fluorescence enzyme immunoassay for insulin (C72, C73). The preparation and properties of “immunomicrospheres”, synthetic polymeric spherical microscopic particles having antibodies chemically bound to their surfaces, have been described, and potential applications to FIA and fluorescence microscopy have been discussed (M32). Principles and practice {of“homogeneous enzyme channeling” fluoroimmunoassay, wherein antigen-antibody binding acts to alter the partitioning of an enzyme between inactive and active microenvironments, have been discussed, and applications of the technique to homogeneous enzyme immunoassays have been reported (M33). A direct fluoroimmunoassay for serum cortisol employs cortisol labeled with fluoresceinthiocarbamyl ethylenediamine and magnetizable cellulose as the solid phase (M34). The magnetizable solid-phase methodology also is employed in a FIA of phenytoin in serum or plasma (M35). A “sequential addition-separation FIJI” for thyroxine in serum also employs a magnetizable solid phase (M36). An immunoassay for steroids is based on the antibody-enhanced hydrolysis of a complex of the steroid with umbelliferone to form fluorescent products. The free steroid inhibits this reaction, and thus attenuates the observed fluorescence, in a concentration-dependentmanner. This is a homogeneous immunoassay requiring no separations (M37). Homogeneous substrate-labeled FIAs for IgG (M38)and IgM (M39) in human serum have been reported; application of the method to other proteins also is discussed (M38). Automation of fluoroimmunoassay procedures has been undertaken (C161, C163). Automated instrumentation for performance of fluorescence polarization immunoassays has been described (14, 15). The possibility that fluoroimmunoassay procedures may be able to distinguish between AI and Az bloodstain subtypes, for forensic purposes, has been discussed (M41). Chemiluminescencce in Immunoassay. The use of chemiluminescence (CIA)measurements in immunoassay has been reviewed (L66,L67). A heterogeneous CL immunoassay for hepatitis B surface antigen uses a two-site solid-phase (“sandwich”)technique, and an isoluminol derivative as the chemiluminescent label (M40). A chemiluminescence immunoassay for estriol-116a-glucuronide in urine has been described. The steroid glucuronide is conjugated with the CL marker aminobutylethylisoluminol, the CL yield of which is enhanced by binding to specific binding protein. This binding, and the concomitant enhancement of CL intensity, is inhibited by the unaltered glucuronide in a concentration-dependent manner (M42). A similar approach iEi used to determine cortisol in dichloromethane extracts of human plasma (M43). A solid-phase CL immunoassay for protesterone in blood plasma has been described; the CL label is a conjugate of progesterone with isoluminol. The analytical figures of merit of this procedure are compared with those for RIA (M44). A CL immunoassay for cortisol proceeds by formation of cortisol conjugates with horseradish peroxidase. The activity of release peroxidase is determined by the luminol-HzOz system following either a first-antibody or double-antibody solid-phase separation (M45). Another immunoassay for cortisol in dichloromethane extracts of plasma uses the CL label aminopentylethylisoluminol covalently bonded to a carboxy derivative of cortisol (M46). A CL competitive-binding assay of human IgG, using luminol, has been described (M47).

FLUOROMETRIC STUDIES OF BIOLOGICAL CELLS, FLOW C’YTOMETRY, AND RELATED T’ECHNIQUES Activity in the study of cells, tissues, and related samples by fluorescence techniques continues at a very high level, and

only a very few of the numerous reports of progress in this area can be cited here. An earlier section in this review dealing with “Sample Illumination Optics” cites a number of microfluorometric techniques of direct relevance to these applications. Instrumentation. Kohen and co-workers have described in detail the instrumentation and techniques for their elegant microfluorometric studies of metabolic processes, transport phenomena, and the intracellular distribution of fluorescent components in biological cells (E6). A scanning inverted mkrofluorometer, which uses electronic shutters to control the illumination and measurrment periods (to minimize photodegradation of fluorochromes),has been described and its use for enzyme activity determinations in single cells has been discussed ( N I ) . An instrument designed for the simultaneous Performance of absorption and fluorescence measurements on biological cells and tissue specimens has been described in detail ( N 2 ) . A 41-chapter monograph on flow cytometry and cell sorting provides an indepth treatment of instrumentation, techniques, arid applications (N3). Instrumentation, techniques, and applications for the quantitation of specific compounds in single biological cells by fluorescence spectrometry have been reviewed by Dolbeare (N4). Methods for presenting and analyzing the data obtained in flow cytometric experiments have been reviewed in detail by Gray and Dean ( N 5 ) . Techniques for extending the dynamic range of fluorescence flow cytometers have been described (N6). Techniques for testing the suitability of optical filters for use in flow cytometry have been described (N7). Modification of a cell sorter to detect simultaneously light of two different wavelengths (from an Ar+ laser) scattered by the cells has been reported, and the utility of this approach in the identification of cell populations is discussed (N8). Although most fluorescence flow cytometric measurements are made at equilibrium, there can be advantages to performing kinetic measurements in conjunction with flow cytometry; techniques and examples of such kinetic measurements have been described ( J 4 ) . Epi-illumination arrangements for performance of polarized fluorescence measurements in flow cytometry have been described (13). Fluorescent Stains for DNA and Other Cellular Components. Lober has prepared a detailed review of the fluorescence of dye-nucleic acid complexes; many of the dyes discussed in this review are those which have achieved prominent analytical use as stains for DNA and RNA (N9). The use of ethidium chloride and ethidium bromide as fluorescent labels for DNA and RNA assays has been reviewed (N36,N37). A comparison of seven common DNA stains has been reported. Also ‘comparedare several different preparative techniques and methods of data analysis. The effects of stain concentration upon flow cytometric results are discussed (NIO). A single-step procedure for staining of DNA in cell nuclei, using conventional stains in a solution also containing a nonionic surfactant, has been described (N11). A subsequent exchange of comments (N12,N13) regarding this method also is of interest. Techniques for “double-staining’’ of cells, for assay of both nuclear DNA and intracellular glycogen, have been described (N14). The use of Hoechst 33662 and propidium iodide as dual DNA stains to discriminate between living and dead cells via flow cytometry has been discussed; discrimination between living and dead cells results from differences in distribution of the two dyes in the cells, and resulting differenceci in the efficiency of energy transfer from 33662 to propidium iodide (N15). Electronic energy transfer phenomena between different fluorescent DNA stains have bleen discussed, and the implications of these phenomena for enhancing staining contrast in flow cytometry have been considered (N26). Errors in flow-cytometricmeasurements of the DNA content of cells, caused by electrolytic degradation of DNA fluorochromes during measurement of electronic cell volume, have been discussed (N17). The possibility that the imprecision of flow cytometric measurements using ethidium bromide and propidium iodide as DNA stains can be decreased by using solvents other than water has been examined; the results indicate that the fluorescence of both dyes is enhanced substantially by use of DzOrather than H20;thus, when DzO is used as solvent, the dye concentration can be decreased substantially and the RSD’s of the measured cell populations are diminished (N18). A comparison of mithramycin and ANALYTICAL CHEMISTRY, VOL. 54, NO. 5, APRIL 1982

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propidium iodide as fluorochromic stains for cellular DNA determinations has been performed. Significant differences in the relative fluorescence signals for the two stains were observed as a function of cell type. These observations reinforce the need for caution in the interpretation of data produced in such assays. It is suggested that differences in the staining behavior of various fluorochromes may ultimately be useful in detecting subtle differences in chromatin structure in different cell populations (N19). The use of propidium iodide as a specific stain for cellular double-stranded RNA in flow cytometry has been discussed; the cells are treated with DNAse prior to staining in order to eliminateinterference from double-stranded DNA (N20). The results of total cellular RNA assays obtained by fluorescence flow cytometry (with acridine orange staining) and UV absorption spectrometry have been compared. The results suggest that staining with acridine orange effectively quantitates total cellular RNA content but that DNA contributes a small fraction of the red “RNA”fluorescence. The intensity of the “green” acridine orange fluorescence correlates with the DNA content of the cells (N21). Factors affecting fluorescence fading in cytofluorometry, and procedures for stabilizing fluorescence of stains, have been discussed (N22). A procedure for localization of specific DNA sequences via fluorescence microscopy is based on covalent binding of the fluorophore tetramethylrhodamine isothiocyanate to the 3’terminus of RNA, which is then hybridized with the complementary DNA. Possible applications of the technique to flow cytometry are discussed (N23). A microassay for DNA employs an enzyme-catalyzed release of desoxyribose from DNA; the liberated desoxyribose reacts with thiobarbituric acid to form a fluorophore. The technique is compared with the ethidium bromide intercalation procedure (”7). An ”enzyme-activated” DNA stain can in principle be produced, in cells,by interaction of a substrate with a specific enzyme to produce a fluorescentproduct which binds strongly to DNA. Such a stain could be used to sort cells on the basis of their enzyme content. The possibility that acridine phosphate can be used as such a substrate (for phosphatase enzymes) has been discussed (N24). The interpretation of the two-peaked DNA distribution frequently observed in the flow cytofluorometric measurement of cellular DNA has been discussed (N30). Experimental conditions for optimizing the “formaldehyde-fluorescent” cytochemical procedure for fluorescent labeling of primary amino groups have been discussed (”5). The possible role of electronic energy transfer phenomena in the apparently anomalously hi h fluorescence intensities produced in the formaldehyde a n f glyoxylic acid histochemical visualization methods for monoamines has been discussed (N29). A technique for cytochemical localization of enzymes in tissue sections, wherein specific binding to the enzyme of an irreversible inhibitor labeled with a fluorophoreis effected, has been described (N26). Criteria for validation of quantitative histochemical techniques for assaying activities of enzymes in single cells and tissue sections have been summarized (NZS). Gluteraldehyde, which is often employed as a fixation reagent for fluorescence microscopy of cells, produces a background fluorescence, the intensity of which is dramatically increased upon exposure of the sample to violet or UV light. Thus, this fixing reagent should if possible be avoided in fluorescence microscopy or else samples fixed with gluteraldehydeshould be used in conjunction with fluorescent probes whose excitation maxima are at relatively long wavelength (N31). Techniques for using formaldehyde as fixative, thereby avoiding the problems associated with gluteraldehyde, have been described (N32). Determination of Specific Substances in Cells and Tissues. A technique for intracellular O2 determination employs pyrene-l-butyrate as the fluorescent probe and a fluorescence video microscope (N33). A modification of this procedure employs pyrene encapsulated in polyacrylamide to form an O2probe with a reported spatial resolution of 0.5 gm (N34).

MISCELLANEOUS TECHNIQUES AND APPLICATIONS Considered in this section are a number of techniques and applications which do not fit comfortably in the preceding sections but appear to be of interest. 144R


Derivative and Modulation Techniques. The effects of scan rate and smoothing on the S I N ratio in derivative spectroscopyhave been considered (01).A circuit for driving an optical modulator has been described (02). Fluorescence Correlation Spectroscopy. A general review of the measurement of instantaneous fluctuations of measured signals from the average response includes a discussion of fluorescence correlation spectroscopy and its application to the study of dynamic chemical equilibria (especially the binding of ethidium bromide to DNA) and to investigations of flowing systems ( 0 3 ) . Applications to Polymer Systems. A procedure for assessment of the compatibility of polymers to formation of blends involves tagging one polymer with an energy donor and the other with an acceptor. The extent to which the chains of the two polymers interpenetrate is estimated by measuring the degree to which energy transfer from the donor to the acceptor occurs, which is straightforward if both species are fluorescent. This procedure is claimed to be more sensitive than conventionalprocedures to small alterations in polymer compatibility ( d 4 ) . Fluorescence depolarization procedures for estimating polymer segment mobilities are discussed (05). The use of pyrene excimer fluorescence as a probe of the rate of end-to-end cyclization in polystyrene has been discussed (06). Instrumentation for measurement of chemiluminescence emitted by polymers undergoing thermo-oxidative reactions has been described and used in simulated studies of polymer aging (07). Other Applications to Clinical and Biochemical Problems. Materials used to clean laboratory glassware may interfere in the fluorometric determination of proteins and amino acids by the o-phthaldialdehydereaction (08). Lamola has reviewed the direct determination of bilirubin and fluorescent porphyrins in blood and other body fluids without prior extraction and has discussed the use of such data in the diagnoses of diseases associated with abnormal or impaired heme synthesis (C112). The use of fluorescence spectroscopy in the structural analysis of polypeptide and protein hormones has been reviewed by Edelhoch and Chen (09). The use of the sensitivity of pyrenebutyric acid to fluorescence quenching by 0 to prepare “oxygen optodes” for determinationof xanthine, factate, and cholesterol has been described. A probe is prepared which contains an inner layer of pyrenebutyric acid and an outer layer containing the appropriate oxydase enzyme. The sample is equilibrated with 0,; as the O2 is consumed by the oxidase (in a manner proportional to the substrate concentration), the pyrenebutyric acid fluorescence intensity is correspondingly enhanced (010). Ethanol in biological fluids is determined by an enzymecatalyzed oxidation of ethanol; the resulting decrease in oxygen concentration is monitored by its quenching of pyrenebutyric acid fluorescence. The detection limit is reported to be ca. 0.01 volume % ethanol (016). A “completely new general approach” to fluorometric enzyme assays, exemplified by use of the fluorogenic substrate methylumbelliferyl 2-acetamide-2-deoxy-P-~-lactosideto determine 6-D-galactosidases with high substrate specificity, has been described (011). The extent to which a sample of DNA enhances the fluorescence of Tb3+ has been reported to depend on the single-strand content of the DNA sample, and conditions for using this phenomenon to measure the extent of “singlestrandedness” in DNA have been described (012). The use of tetracycline to form fluorescent com lexes with various diamagnetic metal cations (e.g., Zn2+,Cdpf, Ba2+)is the basis of a fluorometric method for mapplng distributions of these elements in postmortem human brain samples (013). Techniques for adaptation of the “fluorescamine”reaction system to determination of secondary amines have been described (014). The use of fluorescamine. or o-phthalaldehyde to label N-terminal amino acids of peptides and protelns has been discussed (015). Fluorescent Probes. From among the plethora of references describing the use of fluorescent probes in biological and micellar systems, we can cite only several general reviews. Burns has reviewed general aspects of the use of fluorescent probe techniques in the study of nucleic acids and chromatin in living cells (017). Georghiou has reviewed in detail the study of molecular complex formation in biological systems by fluorometric techniques (018). Eftink and Ghiron have reviewed the use of the quenching of protein fluorescence in


solution by added solutes to make inferences regarding the structure and dynamics of proteins (019). Systematic errors in fluorescence photobleaching recovery experiments (widely used to estimate diffusion rates for fluorophores in cells and membranes) caused by inadequate superimposition of the bleaching and interrogating light images on the sample have been discussed; such errors may be major and are not neceasmily easy to detect because they may be highly reproducible (020). A review by Fendler compares microemulsions, micelles, and vesicles in terms of spectroscopic and photo hysical properties of probes contained therein (021). Ygueragide and Foster have reviewed the fluorescence spectroscopy of biological membranes (022). Kraayenhof has surveyed fluorescent probe techniques for analysis of the architecture of photosynthetic membranes (023). Stryer has reviewed the use of fluorescence probe techniques and Forster resonance energy transfer for estimating proximity relationships in biological macromolecules (024). LITERATURE CITED INTRODUCTION ( A l ) Wehry, E. L. Anal. Ch(em. 1980, 52, 75R-90R. GENERAL BOOKS AND REVllEWS ( B l ) Seitz, W. R. I n “Treatlse on Analytical Chemistry”, 2nd ed. Elvlng, P. J., Meehan, E. J., Kolthoff, I.M., Eds.; Wiley: New York, 1981; Part 1, Vol. 7, pp 159-248. (82) Penzer, G. R. I n “An Introductlon to Spectroscopy for Blochemists”, Brown, S. B., Ed.; Academic Press: New York, 1980; pp 70-114. (83) Wehry, E. L. “Modern Fluorescence Spectroscopy“; Plenum: New York, 1981; Vola. 3 and 4. (84) West, M. A. I n “Specialist Periodical Report Series: Photochemistry”; Bryce-Smith, D., Ed.; The1 Chemical Society: London, 1979; Vol. 10. pp 3-116. (85) Cundall. R. B.; Jones, M. W. I n “Specialist Periodical Report Series: Photochemistry”; Bryce-Smith, D., Ed.; The Chemlcal Society: London, 1979; Vol. 10, pp 117-170. (Be) Bell, J. E. Spectrosc. fllochem. 1981, 7 , 155-94; Chem. Abstr. 1981, 95,57330. (87) Snell, F. D. “Photometric and Fluorometric Methods of Anaiysls: Nonmetals”; Wiley: New York, 1981. (B8) Seitz, W. R. CRC Crl!. Rev. Anal. Clln. Chem. 1980, 8, 367-405. (B9) Zander, M. “Fluorimettie”; Springer: Berlin, 1981. (BIO) HieftJe,G. M.; Travis, J. C.; Lytle, F. E. “Lasers in Chemical Analysis”; Humana Press: Clifton, NJ, 1981. (B11) Robin, M. L.; Schweitzer, G. K.; Wehry, E. L. Appl. Spectrosc. Rev. 1981, 17, 185-88. (812) Lln, S.H. “Radlatlonless Transitions”; Academic Press: New York, 1980. (813) Kemp, T. J. f r o g . Rtiact. Kinet. 1980, 70, 301-98. INSTRUMENTS AND INSTRlJMENTAL COMPONENTS (Cl) Watanabe, Ifi) Yeung, E. S.; Sepaniak, M. J. Anal. Chem. 1980, 52, 1465 A-1481 A. ((271) Yeung, E. S. I n “Lasers In Analytlcal Chemlstry”; Hieftje, G. M., Travis, J. C., Lytle, F. E., Eds.; Humana Press: Clifton, NJ, 1981; pp 273-289. (C72) Hinsberg, W. D., HI; Mllby, K. H.; Zare, R. N. Anal. Chem. 1981, 53, 1509-12. ((273) Lidofsky, S. D.; Hinsberg, W. D., 111; Zare, R. N. R o c . Nat. Acad. Scl. USA 1981, 78, 1901-5. ((274) Strojny, N.; de Sllva, J. A. F. Anal. Chem. 1980, 52, 1554-9. (C75) Strojny, N.; de Silva, J. A. F. I n “Lasers In Chemical Analysis”; HieftJe, G. M., Travis, J. C., Lytie, F. E., Eds.; Humana Press: Clifton, NJ, 1981; pp 225-236. (C76) Yamada, S.; Miyoshl, F.; Kano, K.; Ogawa, T. Anal. Chlm. Acta 1981, 727, 195-8. (C77) Campen, W.; Bachmann, K. Mlkrochlm. Acta 1979, 2, 159-70. fC78) Dina. K.: Kranitzkv, W.: Fischer. S. F.: Kaiser. W. Chem. fhvs. Left. 1980, 72,39-42. . (C79) Wright, J. C. Appl. Spectrosc. 1980, 34, 151-7. (C80) Wlrth, M. J.; Koskelo, A.; Sanders, M. J. Appl. Spectrosc. 1981, 35, 14-21.

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S Immunoassays 80s (Conf.) 1980, 91-112; Chem. Abstr. 1981, 95, 12'8519. (M7) Smith, D. S. UV Spectroin. Group Bull. 1978, 6, 8-16. (M8) Schall. R. F., Jr.; Tenoso. H. J. Clin. Chem. (Winston-Salem. N . C . ) 1981, 27, 1157-84. (M9) Nakamura, R. M.; Dlto, W. R.; Tucker, E. S.,I11 "Immunoassays in the Clinical Laboratory"; Llss: New York, 1979.

(M10) Sternberger, L. A. "[mmunocytochemistry"; Wiiey: New York, 1979. ( M i l ) Tsuji, A. Enzyme Immunoassay 1981, 41-53; Chem. Abstr. 1981, 95,146238. (M12) Boguslaski, R. C.; 1.1, T. M.; Benovic, J. L.; Ngo, T. T.; Burd, J. F.; Carrico, R. C. Lab. Res. Methods Biol. Med. 1980, 4, 45-64; Chem. Abstr. 1981, 94, 167197. (M13) Uiiman, E. F. TokaiS. Exp. Clin. Med. 1979, 4 (Suppi.), 7-32; Chem. Abstr. 1981, 95,76184. (Ml4) Pecht, I.Ann. N . Y . Acad. Sci. 1981, 366, 208-16. (MIS) Wong, R. C.; George, R.; Yeung, R.; Burd, J. F. Clln. Chim. Acta 1980, 100, 65-9. (MI(!) Nguyen, T.-H.; Dockhorn, R. J. Ann. Alergy 1981, 46, 8-11. (MI;') Zaatari, G. S.;Hamilton, S.R.; Jacobs, J.; Datiies, T. 6. Clln. Chim. Acta 1980, 703, 35-7-66, (M10) Ekeke, G. I.; Landon, J.; Edwards, C. R. W.; White, G. W.; Shridi, F. Clin. Chim. Acta 1981, 709,31-7. (MI$)) Li, T. M.; Benovic, J. L.; Buckler, R. T.; Burd, J. F. Clln. Chem. (Winston-Salem, N.C.) 1981, 27,22-8. (M20) O'Donnell, C. M.; McBride, J.; Suffin, S.;Broughton, A. J. Immunoassay 1980, 7 375 -83; Chem . Abstr. 1981, 94,95588. (M21) Aliman, B. L.; Short, F.; James, V. H. T. Clln. Chem. ( Winston-Salem, N.C.) 1981, 27, 1176-!3. (M22) Karnes, H. T.; Gudat, J. C.; O'Donneil, C. M.; Winefordner, J. D. Clin. Chem. (Winston-Salem, N.C.) 1981, 27,249-52. (M23) Wang, R.; Merrili, 13.; Maggio, E. T. Clin. Chim. Acta 1980, 102, 169-77. (M24) Brandtzaeg, P. J. Hktochem. Cytochem. 1981, 29, 1302-15. (M25) Schauenstein, K.; Back, G.; Wick, G. J. Histochem. Cytochem. 1980, 28, 1029-31. (M26) Valnes, K.; Brandtzaeg, P. J. Histochem. Cytochem. 1981, 29, 595-600. (M27) Lim, C. S.; Miiier, J. N.; Bridges, J. W. Anal. Blochem. 1980, 108, 176-84. (M2U) Khanna, P. L.; Uilrnan, E. F. Anal. Biochem. 1980, 708,156-61. (M20) Miller, J. N.; Llm, C. S.;Bridges, J. W. Analyst (London) 1980, 105, 91-2. (M30) Van der Werf, P.; Chang, C.-H. J. Immunol. Methods 1980, 36, 339-47. (M3'1) Rubinstein, K. E. Immunoassays 80s (Conf.) 1981, 127-32; Chem. Abstr. 1981, 95, 128521. (M32) Rembaum, A.; Dreyer, W. J. Science 1980, 208,364-6. (M33) Litman, D. J.; Hanlonl, T. M.; Uiiman, E. F. Anal. Biochem. 1980, 106, 223-9. (M34) Pourfarzaneh, M.; White, G. W.; Landon, J.; Smith, D. S.Clln. Chem. (INinston-Salem, N.C.) 1980, 26,730-3. (M35) Kamei, R. S.;landon, J.; Smith, D. S. Clln. Chem. (Winston-Salem, N.C.) 1980, 26, 1281-4. (M36) Nargessl, R. D.; Ackland, J.; Hassan, M.; Forrest, G. C.; Smith, D. S.; Landon, J. Clln. Chem. (Winston-Salem, N.C.) 1980, 26, 1701-3. (M37) Kohen, F.; Hollander, 2.; Burd, J. F.; Boguslaski, R. C. Res. Steroids 1979, 8 , 147-50. (M38) Ngo, T. T.; Carrico, R. J.; Boguslaski, R. C.; Burd, J. F. J. Immunol. Methods 1981, 42,93-103. (M39) Worah, D.; Yeung, K. K.; Ward, F. E.; Carrico, R. J. Clin. Chert. (INinston-Salem, N.C.) 1981, 27,873-7. (M40) Schroeder, H. R.; Hines, C. M.; Osborn, D. D.; Moore, R. P.; Hurtle, R. L.; Wogoman, F. F.; Rogers, R. W.; Vogeihut, P. 0. Ciin. Chem. (Winston-Salem, N.C.) 1981, 27, 1378-84. (M4I) Liu, J. H.; Klink, F. E.; Nicoi, J. D. J. Forens. Sci. 1980, 25, 686-90. (M421 Kohen, F.; Kim, J. El.; Barnard, G.; Lindner, H. R. Steroids 1980, 36, 405-19. (M43) Kohen, F.; Pazzagli, M.; Kim, J. B.; Lindner, H. R. Sterolds 1980, 36. 421-37. (M44) Kohen, F.; Kim, J. B ; Lindner, H. R.; Collins, W. P. Steroids 1981, 38, 73-88. (M45) Arakawa, H.; Maeda, M.; Tsujl, A. Anal. Blochem. 1979, 97,248-51. (M46) Pazzagli, M.; Kohen, F.; Kim, J. B.; Lindner, H. R. I n "Bioluminescence and Chemiluminescence"; DeLuca, M. A,, McEiroy, W. D., Eds.; Academic Press: New York, 1981; pp 651-7. (M47) Hersh, L. S.;Vann, W. P.; Wilhelm, S. A. Methods Enzymoi. 1981, 73, 608-15. I

FLOW CYTOMETRY AND RELATED TECHNIQUES (NI) de Josselin de Jong, J. E.; Jongkind, J. F.; Ywema, H. R. Anal. Biochem. 1980, 102,120-5. (N2) Bergamini, P. G.; Paimas, G.; Pianteiii, F.; Sani, M.; Cingoiai, M. L.; Leone, L.; Re, L.; Roda, G.; Rossini, L. Chem. Biomed. Environ. Instrum. 1980, IO, 289-309. (N3) Meiamed, M. R.;Mullaney, P. F.; Mendeisohn, M. L. "Flow Cytometry and Sorting"; Wiley: New York, 1979. (N4) Dolbeare, F. A. I n "Modern Fluorescence Spectroscopy"; Wehry, E. L., Ed.; Plenum: New York, 1981; Vol. 3, pp 251-93. (N5) Gray, J. W.; Dean, P. N. Annu. Rev. Biophys. Bioeng. 1980, 9, 509-39. (N8) Hecht, R. M.; Schomer, D. F.; Oro, J. A,; Bartei, A. H.; Hungerford, E. V., I11 J. Hlstochem. Cytochem. 1981, 29,771-4. (N7) Loken, M. R. J. Histochem. Cytochem. 1980, 28, 1136-7. (N8) Loken, M. R.; Houck, D. W. J. Histochem. Cytochem. 1981, 29, 809-15. (N9) Lober, G. J. Luminesc. 1981, 22,221-65. (N10) Taylor, I.W.; Milthorpe, B. K. J. Hlstochem. Cytochem. 1980, 28, 1224-32. ( N i l ) Taylor, I.W. J. Histochem. Cytochem. 1980, 28, 1021-4. (N12) Darzynkiewicz, 2.; Traganos, F.; Meiamed, M. R. J. Hlstochem. Cflochem. 1981, 29,329. (N13,) Taylor, I.J. Histochem. Cytochem. 1981, 29,330. ANALYTICAL CHEMISTRY, VOL. 54, NO. 5, APRIL 1982

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Anal. Chem. 1982, 5 4 , 150R-156R (N14) Tsuchihashi, Y.; Nakanishi, K.; Fukuda, M.; Fujlta, S. Histochemistry 1979, 6 3 , 311-22. (N15) Stoehr, M.; Vogt-Schaden, M. Acta Pafhol. Mlcroblol. Scand. Suppl. 1981, 274, 96-9; Chem. Abstr. 1981, 9 5 , 93328. (N16) Latt, S. A.; Sahar, E.; Eisenhard, M. E.; Juergens, L. A. Cytomefry (Baltimore) 1980, 1 , 2-12; Chem. Absfr. 1981, 9 5 , 2931. (N17) Alabaster, 0.; Glaublger, D. L.; Harnllton, V. T.; Bentley, S.A,; Shackney, s. E.; Skramstad, K. s.; Chen, R. F. J. Histochem. Cytochem. 1980, 2 8 , 330-4. (N18) Mazzini, 0.; Giordano, P. Acta Pathol. Microbiol. Scand. Suppl. 1981, 274, 74-7; Chem. Abstr. 1981, 9 5 , 57554. (Nl9) Hamllton, V. T.; Habbersett, M. C.; Herman, C. J. J. Hlstochem. Cytochem. 1980, 2 8 , 1125-8. (N20) Frankfurt, 0. S. J . Hlsfochem. Cytochem. 1980, 2 8 , 493-8. (N21) Bauer, K. D.; Dethlefsen, L. A. J . Hisfochem. Cytochem. 1980, 2 8 , 493-8. (N22) Fukuda, M.; Tsuchihashl, Y.; Takamatsu, T.; Nakanishi, K.; Fujita, S. Hlstochemlsfty 1980, 6 5 , 269-76. (N23) Bauman, J. G. J.; Wiegant, J.; Van DuiJn, P. J . Hlsfochem. Cytochem. 1981, 2 9 , 238-46. (N24) Tsou, K. C.; Yip, K. F.; Miller, E. E. J . Histochem. Cytochem. 1980, 2 8 , 1032-6. (N25) Hougaard, D. M.; Larsson, L. I.Hlsfochemistry 1981, 72, 401-13. (N26) Gilad, G. M.; Gilad, V. H. J . Hlstochem. Cytochem. 1981, 29, 687-92. (N27) Nordling, S.; Aho, S. Anal. Blochem. 1981, 775, 260-6. (N28) Stoward, P. J. I n “Trends in Enzyme Histochemistry and Cytochemlstry” (Ciba Foundation Symp 7 3 ) ; Excerpta Medlca: Amsterdam, lb80; pp 1-5. (N29) Lindvall, 0.; Bjoerklund, A,; Falck, B.; Loren, I. Hlstochemistry 1980, 68, 169-81. (N30) Wood, J. C. S.; Todd, P. Cell. Biophys. 1979, 7, 211-8; Chem. Abstr. 1980, 9 2 , 90297. (N31) Colllns, J. S.; Goldsmith, T. H. J . Hlstochem. Cytochem. 1981, 2 9 , 41 1-4. (N32) Berod, A.; Hartman, B. K.; Pujol, J. F. J . Hlstochem. Cytochem. 1981, 2 9 , 844-50. (N33) Benson, D. M.; Knopp, J. A,; Longmulr, I.S. Blochlm. Biophys. Acta 1980, 597, 187-97. (N34) Podgorski, G. T.; Longmulr, I. S.; Knopp, J. A,; Benson, D. M. J. Cell. Physiol. 1981, 707,329-34. (N35) Luebbers, D. W.; Voelkl, K. P.; Grossmann, U.; Opitz, N. Prog. Enzyme Ion-Sel. Electrcdes (Proc . Meet. Theory Appl. Ion-Sel. Electrodes

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Physiol. Med.) 1981, 67-73; Chem. Abstr. 1981, 9 5 , 128592. (N36) Morgan, A. R.; Lee, J. S.; Pulleyblank, D. E.; Murray, N. L.; Evans, D. H. Nucleic Acids Res. 1979, 7 , 547-69. (N37) Morgan, A. R.; Evans, D. H.; Lee, J. S.; Pulleybank, D. E. Nucleic Acids Res. 1979, 7 , 571-94.

MISCELLANEOUS TECHNIQUES AND APPLICATIONS (01) O’Haver, T. C.; Begley, T. Anal. Chem. 1981, 5 3 , 1876-8. (02) Hawthorne, A. R. Rev. Sci. Instrum. 1980, 5 7 , 1275-6. (03) Doherty, J. V.; Clarke, J. H. R. Sci. Prog. (Oxford) 1980, 66, 385-419. (04) Amranl, F.; Hung, J. M.; Morawetz, H. Macromolecules 1980, 73, 649-53. (05) Elmgren, H. J. Polym. Sci., Polym. Lett. Ed. 1980, 78,351-6. (06) Winnik, M. A.; Redpath, T.; Richards, D. H. Macromolecules 1980, 73, 328-35. (07) Fanter, D. L.; Levy, R. L.; Llppoid, K. 0. Org. Coat. Plast. Chem. 1978, 3 9 , 603-6; Chem. Abstr. 1980, 9 3 , 115309. ( 0 8 ) Shute, D. J. Med. Lab. Sci. 1980, 3 7 , 173-4. (09) Edelhoch, H.; Chen, R. F. Horm. Protelns Pept. 1980, 9 , 109-73. (010) Voelkl, K. P.; Grossmann, U.; Optiz, N.; Luebbers, D. W. Adv. Physiol. Sci., Proc. Int. Congr., 28th 1981, 2 5 , 99-100; Chem. Abstr. 1981, 9 5 , 20764. (011) Creme, S.;Leaback, D. H. Anal. Biochem. 1980, 103, 258-63. (012) Ringer, D. P.; Howell, B. A.; Kizer, D. E. Anal. Blochem. 1980, 703, 337-42. (013) MacInnes, D. G.; Reading, H. W.; Glen, A. I.M. Biochem. SOC. Trans. 1980, 8 , 340-1. (014) Nakamura, H.; Tamura, 2 . Anal. Chem. 1980, 5 2 , 2087-92. (015) Mendez, E.; Marco, R. Methods Pepf. Protein Sequence Anal., Proc. Int. Conf., 3rd 1980, 295-308; Chem. Abstr. 1981, 9 4 , 60924. (016) Voelkl, K. P.; Opitz, N.; Luebbers, D. W. Fresenlus’ 2.Anal. Chem. 1980, 307, 162-3. (017) Burns, V. W. Photochem. Photobiol. Rev. 1980, 5 , 87-103. (018) Georghiou, S.I n “Modern Fluorescence Spectroscopy”; Wehry, E. L., Ed.; Plenum: New York. 1981; Vol. 3, pp 193-249. (019) Eftink, M. R.; Ghiron, C. A. Anal. Blochem. 1981, 774, 199-227. (020) Barlsas, B. G. Biophys. J . 1980, 2 9 , 545-8. (021) Fendler, J. H. J . Phys. Chem. 1980, 8 4 , 1485-91. (022) Yguerabide, J.; Foster, M. C. I n “Membrane Spectroscopy”; Grell, E., Ed.; Springer: Berlln, 1981; pp 199-269. (023) Kraayenhof, R. Mefhods Enzymol. 1980, 6 9 , 510-20. (024) Stryer, L. Annu. Rev. Biochem. 1978, 47, 819-46.

X-ray Spectrometry G. L. Macdonald Central Materials Laboratory, Mullard Mitcham, Surrey, England CR4 4XY

The most fascinating aspect of X-ray spectrometric development in the period under review (late 1979 to late 1981) is the ingenuity with which a steadily widening range of primary beams, manipulated in various ways, is being used to bombard an even wider range of samples. The detector is almost, but not quite, without exception the energy-dispersive spectrometer with a liquid nitrogen cooled Si(Li) detector a t its heart, and there is still no real sign of this invention of the late 1960s being superseded. The new class of techniques labeled AEM (1-5) (analytical electron microscopy) has X-ray spectrometry as a dominant partner and the newer uses of synchrotron sources include X-ray analysis although at present the emphasis is on EXAFS (6)(extended X-ray absorption fine structure) rather than on more conventional X-ray emission spectrometry (7). The other dominant feature of the period is the continuity of effort toward improved quantitation. The ready availability of low-cost laboratory microcomputers has led to some simplification of the software available for X-ray work. Most of the useful work is, however, a tidying up of loose ends, the best feature of which is a coming together of the “fundamental parameter” and the “empirical coefficient” schools with the recognition that, as usual, a compromise offers the best solution. Before expanding on this quantitative theme, however, it is more sensible to revert to discussion of developments in the generation, detection, and general instrumentation for 150 R

0003-2700/82/0354-150R$06.00/0

X-rays in the spectrometric field.

EXCITATION Particle Induced X-ray Emission (PIXE). The relatively new branch of X-ray spectrometry labeled PIXE by those who practice it tends to grow separately because the high-energy particle sources (by common consent, electrons are excluded) are few in number (probably fewer than 100 (8)) and its development literature tends to appear in the nuclear journals rather than the analytically oriented ones. For these reasons it is sensible to deal separately with the topic in this review. Early work was largely concerned with the excellent concentration sensitivity of PIXE, particularly for the heavier elements, but some emphasis in recent years has been placed on reducing the irradiated area and providing a competitor to EPMA (electron probe microanalysis) by using particle (usually proton) probes, and hence improving the mass sensitivity. Cahill (8) in a recent review lists 9 collimated ion probes and 19 focused beam probes. Legge (9)makes a careful comparison of proton and electron microprobes, and Doyle et al. (10) give additional information about other useful interactions between beam and sample. Aronson and Horowitz (11)describe a scanning proton microprobe, as do Chuang et al. (12). A somewhat different approach, but unique to PIXE in the information that can be obtained, is that of scanning 0 1982 American Chemical Society

Molecular fluorescence, phosphorescence, and chemiluminescence

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