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(100) Ito, S.:Haraguchi. K.; Nakagawa, K.; Yamada, K. BunsekiKagaku 1977, 26, 554-559; Chem. Abstr. 1978, 8 8 , 163250n. (101) Ito, S.; Haraguchi, K.; Nakagawa, K. Bunseki Kagaku 1978, 27, 334338; Chem. Abstr. 1979, 9 0 , 33421. (102) Stepanov, A. V.; Nikitina, S. A.; Dem'yanova, T. A. Radiokhimiya 1979, 21,34-38; Chem. Abstr. 1979, 91, 13085k. (103) Stepanov, A. V.; Nemtsova, M. A,; Nikitina, S. A,; Dem'yanova, T. A. Radiokhimiya 1978, 20, 906-910; Chem. Abstr. 1979, 9 0 , 161628j. (104) Albrecht-Gary, A.-M.; Collin, J.-P.; Jost, P.; Lagrange, P.; Schwing, J.-P. Analyst (London) 1978, 103,227-232. (105) Lagrange. J.; Lagrange, P.; Zare, K. Bull. SOC.Chim. f r . 1978, I( 1-2). 7-16. (106) Kitagawa, T.; Fujikawa, K. Nippon Kagaku Kaishi 1977, 7 , 998-1002; Chem. Abstr. 1978, 8 8 , 57938h. (107) Pelizetti, E.; Giraudi, G.; Mentasti, E. Anal. Chim. Acta 1977, 9 4 , 479-483. (108) Aksel'rud, G. A.; Yaremchuck, B. N. Zh. Prikl. Khim. 1978, 57, 2213-2217; J . Appl. Chem. U . S . S . R . (Engl. Trans/.) 1979, 51. 2 108-21 1 1. (109) Hiraki, K.; Morishige, K.; Nishikawa, Y. Anal. Chim. Acta 1978, 97, 12 1- 128. (110) Onoue. Y.; Morishige, K.; Hiraki, K.; Nishikawa, Y. Anal. Chim. Acta 1979, 106,67-72. (111) Pelizzetti, E.; Mentasti, E. Anal. Chim. Acta 1979, 108, 441-443. (112) Shapenova, G. Kh.; Talipov, Sh. T.; Orlik, I. A, Uzb. Khim. Z h . 1978, 4 , 8-13; Chem. Abstr. 1978, 8 9 , 1 4 1 7 0 0 ~ . (113) De Oliveira, W. A.; Rodella, A. A. Talanta 1979, 26, 965-967. (114) Koupparis, M. A.; Efstathiou, C. E.; Hadjiioannou, T.P. Anal. Chim. Acta 1979, 107, 91-100. (115) Lazarou, L. A.; Siskos, P. A.; Koupparis, M. A,; Hadjiioannou, T. P.; Appelman, E. H. Anal. Chim. Acta 1977, 9 4 , 475-478. (116) McCracken, M. S.;Malmstadt, H. V. Talanta, 1979, 26, 467-472. (1 17) Kreingol'd, S. U.; Kefilyan, L. I.; Antonov, V. N. Zh. Anal. Khim. 1977, 32, 2424-2428; Chem. Abstr. 1978, 8 8 , 182076b. (118) Altinata. A.; Pekin, B.; Ulgu, S. Ana/yst(London) 1977, 102,876-878. (119) Steinhart. H. Anal. Chem. 1979, 57, 1012-1016. (120) Kreingol'd, S. U.; Antonov, V. N.; Yutal, E. M. Z h . Anal. Khim. 1977, 32, 1618-1623; Chem. Abstr. 1978, 8 8 , 784932. (121) Goulden, P. D.; Anthony, D. H. J. Anal. Chem. 1978, 5 0 , 953-958. (122) Hiromi, K.; Fujimori, H.; Yamaguchi-ito, J.; Nakatani, H.; Onishi, M.; Tonomura. B. Chem. Lett. 1977, 1333-1336. (123) Mudretsov, A. I. Tekhnol. Drev. Plit. Plast. 1977, 4 , 66-71; Chem. Abstr. 1979, 90, 249781.
(124) Yamashita, M.; Marugama, T.; Komatsu, W. Z . Phys. Chem. (Wiesbaden) 1977, 105, 187-196; Chem. Abstr. 1977, 8 7 , 157486g. (125) Slais,K.; Krejci, M. J . Chpmatogr. 1978, 148, 99-110. (126) Krejci, M.; Slais, K.; Tesarik, K. J . Chromatogr. 1978, 149, 645-652. (127) Holcombe, J. A.; Eklund, R. H.; Smith, J. E. Anal. Chem. 1979, 51. 1205-1209. (128) Carter, T. J. N.; Stanbridge, B. R. Ana/yst(London) 1978, 703,968-972. (129) Tawa, R.; Hirose, S. Talanta 1979, 26, 237-243. (130) Montano, L. A.; Ingle, Jr., J. D. Anal. Chem. 1979, 5 1 , 919-926. (131) Montano, L. A.; Ingel, Jr.. J. E. Anal. Chem. 1979, 51. 926-930. (132) MacDonald, A.; Chan, K. W.; Nieman, T. A. Anal. Chem. 1979, 51, 2077-2082. (133) Veazey, R. L.; Nleman. T. A. Anal. Chem. 1979, 5 1 , 2092-2096. (134) Yap, W. T.; Cummings, A. L.; Margolis, S. A.; Schaffer, R . Anal. Chem. 1979, 5 1 , 1595-1596. (135) Connors, K. A.; Pandit, N. K. Anal. Chem. 1978, 5 0 , 1542-1545. (136) Weisz, H.; Pantel, S.; Giesin, R. Anal. Chim. Acta 1978, 101, 187-191. (137) Weisz, H. Anal. Chim. Acta 1964. 30, 163-166. (138) Mieling, G. E.; Pardue, H. Anal. Chem. 1978, 50, 1611-1618. (139) Schwartz, L. M.; Gelb, R. I. Anal. Chem. 1978, 50, 1592-1594. (140) Carr, P. Anal. Chem. 1978. 50, 1602-1607. (141) Kelter, P. B.; Carr, J. D. Anal. Chem. 1979, 51, 1825-1828. (142) Kelter, P. B.; Carr, J. D. Anal. Chem. 1979, 51, 1828-1834. (143) Kelter, P. B.; Carr, J. D. Anal. Chem. 1979, 57, 1857. (144) Mottola. H. A.; Hanna. A. Anal. Chim. Acta 1978, 100, 167-180. (145) Davis, J. E.; Pevnick, J. Anal. Chem. 1979, 51, 529-533. (146) Fox, Jr., J. B. Anal. Chem. 1979, 51. 1493-1502. (147) Goulden, P. D.; Anthony, D. H. J. Anal. Chem. 1978, 5 0 , 953-958. (148) Zsakb, J. Anal. Chem. 1978, 5 0 , 1105-1107. (149) Bunzl, K. Anal. Chem. 1978, 5 0 , 258-267. (150) Cammann, K. Anal. Chem. 1978, 5 0 , 936-940. (151) Cox, J. A.; Cheng, K.-H. Anal. Chem. 1978, 5 0 , 601-602. (152) Caserta, K. J.; Holler, F. J. Crouch, S. R.; Enke, C.G. Anal. Chem. 1978, 50, 1534-1541. (153) Holler, F. J.; Crouch, S. R.; Enke, C. G. Chem. Instrum. 1977, 8 , 111-1 19. (154) Mieling, G. E.; Pardue. H. L. Anal. Chem. 1978, 50. 1333-1337. (155) Whiting, L. F.; Carr, P. W. Anal. Chem. 1978. 5 0 , 1997-2006. (156) Gulberg, E. L.; Christian, G. D. Chem., Biomed.. andEnviron Instrum. 1979, 9 . 277-281. (157) Karweik, H.; Huber, C. 0. Anal. Chern. 1978, 5 0 , 1209-1212. (158) Ratzlaff, K. L.; Chung, F. S.;Natusch, D. F. S.; O'Keefe, K. R. Anal. Chem. 1978, 50, 1799-1804.
Electron Microscopy Michael Beer The Department of Biophysics, Johns Hopkins University, Baltimore, Maryland 2 12 18
Biological electron microscopy is a vast and enormously active area of research. I have concentrated in this review on technical aspects of structure determination which may have broad impact on the field. No attempt was made to review particular areas of biology unless in those areas approaches were used which promise to have wide applicability. The period covered is 1978 to December 1979.
LOW D O S E ELECTRON MICROSCOPY Electron microscopy of biological samples is limited today not so much by the resolution of the instruments as by the damage which inevitably accompanies irradiation. In 1975 Unwin and Henderson (1) published a classic paper in which they showed how for a two-dimensional crystalline array this limitation can be circumvented by spreading the total electron dose necessary for high resolution over a large number of unit cells and then by image processing obtaining a single composite high resolution image of the unit cell. They studied the naturally crystalline membrane of the purple bacterium Halobacterium halobium. These low dose procedures are of course applicable to any material which can exist as a two-dimensional crystal. Accordingly Chiu and Glaeser ( 2 ) are studying the structure of the gene 32 protein, GP32. The attraction of the procedure has now been recognized by many workers and several systems are under investigation ( 3 ) . Unwin and Henderson ( I ) in their original paper recognized that their electron diffraction patterns indicated specimens 40 R
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ood to about 3 A, yet the images obtained had resolution no %etter than 7 A. This discrepancy is probably due to instrumental distortions and aberrations. Perhaps the most important of these is variation in magnification over the large fields generally observed, which leads to barrel or pincushion distortion. T o avoid loss of structural information manufacturers will have to design microscopes such that the magnification is constant over the field.
LOW TEMPERATURE MICROSCOPY Not all biologically interesting entities can be incorporated into two-dimensional arrays. For those which resist crystallization either prior to observation or during exposure, damage can apparently be reduced by various low temperature procedures. Low Temperature of Specimen Reduces Beam Dependent Artifacts. The studies of Unwin and Henderson ( I ) showed clearly that to avoid beam damage, dosages had to be kept below about 0.5 e A2. Hayward and Glaeser ( 4 ) , using the same membrane, s owed that if the specimens are cooled to about -120 OC the structure will tolerate some 3-7 times greater irradiation than a t room temperature. Similar results were obtained by Taylor and Glaeser (5) on frozen crystals of catalase. For this type of microscopy manufacturers do offer specimen stages with which the sample can be cooled to near the temperature of liquid nitrogen. Unfortunately the commercial ones are still unstable, whereas those modified to give better
h
0 1980 American
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ELECTRON MICROSCOPY
Mlchael Beer is professor of biophysics at Johns Hopkins University. He was born in Budapest, Hungary, but moved to Canada in 1938. He received a B.A. in physics and chemistry in 1949 and an M.A. in 1950 from the University of Toronto and earned his Ph.D. in physical chemistry at the University of Manchester, England, in 1953. He was a research associate in physics at the University of Michigan from 1953 to 1954 when he became an assistant professor in physics. After serving as a Fellow of the National Research Council of Canada, Ottawa, from 1956 to 1958, he joined the faculty at Johns HoDkins Universitv. Dr. Beer is a oast DresMicroscopy Society of America
thermal insulation and a symmetrical arrangement of supports will allow resolution considerably better than 10 A (6).It will be important to improve the commercial design to give sufficient stability for high resolution. Spectacular preservation of specimens cooled to liquid helium temperatures was reported by Fox, Knapek, and Weyl (7), who used the Siemens electron microscope with a superconducting lens described by Dietrich (8). These preliminary results have now been extended by Dubochet and Knapek (9) who found that the radiation resistance of organic materials a t the temperature of liquid helium is greater than at room temperatures by around two orders of magnitude. The above results were based on measurements of the electron dosages which led to the disappearance of diffraction spots observed with organic crystals. The results are consistent with the earlier results of Ramamurti et al. (10) and Dubochet ( I I ) , which indicated that a t liquid helium temperatures electron irradiation does not lead to mass loss. The importance of these results is that they hold out the exciting possibility that if the specimen is cooled to the temperature of liquid helium, its survival during microscopy may be adequate for high resolution. A second beam dependent specimen change which has long plagued electron microscopists is contamination. This results from the beam reaction of hydrocarbons with the specimen. These are either adsorbed from the relatively poor vacuum found in many instruments or are brought in by the specimen. The former source has largely been eliminated by improved vacuum systems and cold surfaces near the specimen. The latter source of contamination, as recently shown by Voreades and Wall (12), is eliminated by even moderate cooling of the specimen to -60 “C. Presumably a t the lower temperature the adsorbed hydrocarbons do not diffuse a t appreciable rates into the irradiated region. Rapid Freezing t o Preserve Structure. It is well known that rapid freezing of biological specimens avoids many of the artifacts of chemical fixation. Also it is recognized that maximum rate of freezing requires a coolant which does not boil on contact with the warm tissue, for otherwise an insulating vapor layer would form and prevent rapid heat exchange. For this reason isopentane and certain fluorocarbons have been used. In the early sixties Van Harreveld and Crowell (13) introduced a cold copper block to freeze specimens suddenly pressed against it. In recent times this approach has seen some important developments and applications. Heuser et al. (14) used the vapor of liquid helium to cool a copper block to near 4 K. In addition these workers were able to measure the time required to freeze the tissue by following the change in dielectric properties as the aqueous sample freezes. It was found that in 1 or 2 ms the surface layers froze; also the time of freezing a t various depths in the tissue could be determined. Heuser et al. (14) used this approach to follow the emptying of the vesicles containing neurotransmitters in nerve terminals. This is but one important example of how the kinetics of morphological changes in the millisecond range might be followed for a variety of biological systems.
ELECTRON ENERGY LOSS SPECTRA Electron energy loss spectra have long been considered a potentially powerful tool for the identification of atoms and molecules. During the past few years the problems and op-
portunities have been better defined and for the first time this approach has begun to see some important applications in biology. Two types of instruments have been used to obtain energy loss spectra: (1)spectrometers coupled to STEM or CTEM and (2) CTEM provided with a Castaing-Henry energy filter. In one important application, Costa et al. (15) estimated the abundance of serotonin in biological tissue by using instead of the normal form a fluorine-containing active analogue and determining its abundance through the characteristic energy loss spectrum of fluorine a t 680 V. In the experiments reported, a probe resolution of 10 nm was used and in three successive exposures the signal did not diminish suggesting that fluorine was not lost. I t was estimated that 2 X g of fluorine were detected, a number which will almost certainly further decrease as smaller probes and low temperature s for the reduction of mass loss become available. Conceiva ly in the near future even individual atoms will be recognized by electron energy loss spectra! Using a CTEM with a Castaing-Henry filter Simon and Ottemsmeyer (16) suggested that phosphorus-containing components could be recognized through the characteristic 150-eV loss.
??
DETERMINATION OF MASS OR MOLECULAR WEIGHT Zeitler and Bahr (17) as early as 1962 recognized that the mass of an object is directly reflected in the number of electrons scattered by it during electron microscopic examination. In those days mass measurements required the calibration of the density of photographic plates as a function of electron exposure and the densitometry of micrographs, a laborious and often inaccurate procedure. The high performance STEM instruments today are coupled to computers. With such instruments the operations involved in the determination of mass or molecular weight are very simple. Thus these instruments are becoming perhaps the most direct means for evaluating molecular weights of biological objects. The procedures to be used have been described by Engel (18) and by Wall (19); an interesting application to the structure of nucleosomes has been published by Woodcock e t al. (20).
ELECTRON MICROSCOPY OF LABELED SYSTEMS The successful imaging of the larger atoms was first accomplished by Crewe et al. (21) using a STEM and soon afterwards by Henkelman and Ottemsmeyer (22) using a CTEM in dark field mode. A recent symposium (23) on “Direct Imaging of Atoms in Crystals and Molecules” summarizes the progress in this area since that time. In molecular biology this instrumental capability suggested the attractive possibility that chemical groups or particular macromolecules might be identified by appropriate selective labeling with heavy atoms. The discovery that heavy atoms are mobile on the surfaces of support films (24) raised questions about the precision of these experiments. Apparently the movement is quite variable. Langmore e t al. (25) found that Hg atoms when used as labels for certain bases in nucleic acids under the conditions required for their imaging moved extensively and sometimes coalesced into larger clusters. Similar results with Hg atoms bound to fd virus were found by Lipka et al. even when the specimen stage was cooled to -120 “C (26). On the other hand Cole e t al. (27), who examined poly U with every base labeled with an Os atom, observed these heavy atoms in a configuration which suggested they had moved little-probably less than 10 &after they were deposited on the support films. There is some evidence that Pt atoms also are less motile than Hg and may be suitable as heavy atom labels (28). It is worth mentioning that Stewart and Diakiw (29) synthesized a thiol reagent with four mercury atoms and used i t successfully to orient the monomers in paracrystals of tropomyosin. However they studied aggregates of molecules probably using lower dosages than would be required for single atom imaging. Thus their results do not contradict the view that Hg is an unstable marker while osmium and platinum are more stable. Several reactions have now been developed for binding heavy atoms to particular chemical groups. Thus Os atoms can be bound to the pyrimidines of nucleic acids (30). Or if ANALYTICAL CHEMISTRY, VOL. 52, NO. 5, APRIL 1980
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the nucleic acid is first modified with chloracetaldehyde to give etheno derivatives with adenine and cytosine, then each adenine residue will also bind an Os atom (31). An elegant procedure for roducing double stranded DNA with particular nucleotides lateled was introduced by Strothkamp and Lippard (32) who incorporated phosphorothioate groups into DNA in an enzymatic synthesis and showed the sulfur-containing nucleotides specifically bound to platinum atoms. These reactions allow specific labeling protocols for various groups of bases in nucleic acids and promise to be useful in establishing sites of binding of proteins. For labeling proteins the compound (Gly-L-Met) PtCl has been shown to bind only to methionine residues in collagen and electron micrographs show reaction at the sites expected from the known methionine positions (33). A similar specificity for methionine was found by Dumont and Wiggins who studied the reactions of this reagent in the membranes of the purple bacterium Halobacterium halobium (34). Finally it has recently been shown that certain Os(V1)ligand complexes react with sugar residues. Again electron micrographs or aggregates of the glycoprotein rat tail tendon collagen showed density only where amino acid sequence analysis established the sites of glycosylation (33). Use of the above reagents is a t an early stage. Yet when used in conjunction with microscopes able to image the larger atoms, they show considerable promise in the localization of various chemical groups within macromolecules or cells. LITERATURE CITED
(1) Unwin, P. N. T.; Henderson, R. J. Mol. Bid. 1975, 94, 425-440. (2) Chiu, W.; Glaeser, R. M. J. Mol. Biol. 1978, 122, 103-107. (3) “Regular 2-D Array of Biomacromolecules: Structure Determination and Assembly”; workshop held in Burg Gemen, Germany, June 17-21, 1979; Springer-Verlag: West Berlin and Heidelberg, 1979, to be published. (4) Hayward, S. B.; Glaeser, R. M.; Ultramicroscopy 1979, 4 , 201-210., (5) Taylor, K. A,; Glaeser, R. M.; J. Uffrastruct. Res. 1976, 55, 448. (6) Hayward, S. B.; Glaeser, R . M. Ufframicroscopy, in press.
(7) Fox, F.; Knapek, E.; Weyl, R. Electron Microsc., Proc. Int. Congr., 9th, 1978. 1978. 2. 342-343. (8) Dietrich, I. Electron Microsc., Proc. Int. Congr., 9th, 1978, 1978, 3 ,
173- 184. (9) Dubochet, J.; Knapek, E. Chem. Scr. 1978-1979, 14, 267-269. (10) Ramarnurti, K.: Crewe, A. V.; Isaacson. M. S. Ultramicroscopy1975, I . 156-1 58. (11) Dubochet, J. J . Uffrastruct. Res. 1975, 52, 276-268. (12) Voreades, D.; Wall, J. S. 37th Annual EMSA Meeting, San Antonio, 1979, pp 358-359. (13) Van Harreveld, A.; Crowell, J. Anat. Rec. 1900, 19. (14) Heuser, J. E.; Reese, T. S.; Dennis, M. J.; Jan, Y.; Jan, L.; Evans, L. J. Cell. B i d . 1979, 81, 275-300. (15) Costa, J. L.; Joy, D. C.; Mahr, D. M.; Kirk, K. L.; Hui, S. W. Science 1978, 200, 537-539. (16) Simon, G. T.; Ottemsmeyer, F. P. Thirty-seventh Annual EMSA Meeting, San Antonio. 1979. DD 510-511. (17) Zeitler, E.; Bahr,’G.-F. J. Appl. Phys. 1962, 33, 847. (18) Engel, A. Ufframicroscopy 1978, 3, 273-281. (19) Wall, J. S. Scanning Electron Microsc. 1979, 2 , 291-302. (20) Woodcock, C. L. F.; Fredo, L.-L. Y.; Wall, J. S. J. Cell. Biol. 1979, 83, 156a. (21) Crewe. A. V.; Wall, J.; Langmore, J. Science 1970, 168, 1338-1340. (22) Henkelrnan. R. M.; Ottemsmever. P. F. Proc. Nat. Acad. Sci. USA 1971, 68, 3000-3004. (23) Kihlbong, L.. Ed.; Chem. Scr. 1978-1979, 14, 1-295. (24) Isaacson, M.; Kopf, D.; Uthnt, M.; Parker, N. W.; Crewe, A. V. Proc. Ut/. Acad. Sci. USA 1977. 7 4 . 1802-1806, (25) Langmore, J. P.; Crewe,’ A. V. 32nd Annual EMSA Meeting, St. Louis, 1974, pp 376-377. (26) Lipka J. J.; Lippard, S.J.; Wall, J. S. Science 1977, 206, 1419-1421. (27) Cole, M. D.; Wiggins, J. W.; Beer, M. J. Mol. Biol. 1977 117, 387-400. (28) Germinario, L. T.; Reed, R.; Cole, M. D.; Rose, S. D.: Wiggins, J. W.; Beer, M. Scanning Nectron Microsc. 1978, 1 , 69-76. (29) Stewart, M.; Diakiw, V. Nature(London) 1978, 274, 184-186. (30) Chang, C.-H.; Beer, M.; Marzilli, L. Biochemistry 1977 16, 33-38. (31) Marzilli, L. G.; Hanson, B. E.; Kapili. L.; Rose, S. D.; Beer, M. Bioinorg. Chem. 1978, 8, 531-524. (32) Strothkamp, K. G.; Lippard, S. J. Proc. Natl. Acad. Sci. USA 1976, 73, 2536-2540. (33) Beer, M.; Wiggins, J. W.; Alexander, R.; Schettino, R.; Stoeckert, C.; Piez, K. A. 37th Annual EMSA Meeting, San Antonio, 1979, pp 28-29. (34) Dumont, M. E.; Wiggins J. W. J . Supramol. Struct. Suppl. 3 1979, 114.
Organic Elemental Analysis T. S. Ma” Department of Chemistry, City University of New York, Brooklyn, New York 11210
Mllton Gutterson Flavor Application Laboratory, Dragoco Inc., King Road, Totowa, New Jersey 075 12
GENERAL This review follows the previous one (1) and covers the literature from December 1977 to September 1979. The reader is referred to a recently pubIished comprehensive treatise on organic elemental analysis (2) in which the senior author described a number of unpublished methods that are in operation in various laboratories. These methods will not be cited in the present review. After the normalization of U. S.-China relations in 1979, the senior author went to China for a three-month lecture tour and visited more than 30 research institutions in 12 cities. It was noted that there were many innovations in organic microanalysis and several automated devices were constructed in China (3),but most of the research work has not been published. A major development in the large organic analytical laboratories during recent years ( 2 ) is concerned with the computerization and data processing of elemental analyses that are amenable to total automation. Some programs have been evaluated. Thus Bramstedt and Harrington ( 4 ) published 42 R
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results indicating that a C-H-N system can be automated and computerized for increased flexibility while maintaining a high degree of accuracy and precision. van den Bosch et al. ( 5 ) developed a microcomputer for data processing of C-H-N and 0, C-H-N and S, or 0 and S. Brodkorb et al. (6) described analyzers for C, H, S, and halogens; readings are classified and data are printed out by computer, inconsistent results being signaled by an alarm device. Merz (7) discussed the rapid determination of C, H, N, 0, S, and halogens and pointed out some limitations of these methods. Eastin (8) described an automated spectrophotometric finish of semimicro-Kjeldahl analysis with results similar to those obtained by distillation and titration. Nowadays most organic materials submitted for analysis are mixtures (9). For the determination of the elements in these materials, improvements of the techniques for the destruction of organic matter were studiously investigated, such as the Kjeldahl digestion procedures (10-14). Erni and MWer (15) worked out a mathematical model for the optimization
0 1980 American
Chemical Society
