Characterization of Organic Compounds - ACS Publications

R. H., Ind. Eng. Chem., Anal. Ed., 15, 287 (1943). (321) Thomas, M. D., I vie, J. O., and Fitt, T. C., Ibid., 18, 383. (1946). (322) Thomas, P. R., Do...
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V O L U M E 2 2 , N O . 1, J A N U A R Y 1 9 5 0 (316) Taylor, H. F., Gas J., 252, 293 (1947). (317) Taylor, R. C., and Young, W. S., IND.ENG.CHEM.,. ~ N A L .E D . , 17, 811 (1945). (318) Thiel, C. C., Hills G. L., and Scharp. L. R., Dairy Research, 15, 121 (1947). (319) Thomas, H. H., and Cooper, J., Gas World, 128, 182 (1948). (320) Thomas, M. D., Ivie, J. O., Abersold, J. N., and Hendricks, R. H., IND.ENG.CHEM.,ANAL.ED., 15, 287 (1943). (321) Thomas, M. D., Ivie, J. O., and Fitt, T. C., Ibid., 18, 383 (1946). (322) Thomas, P. R., Donn, L., and Becker, H. C., ANAL.CHEM., 20, 209 (1948). (323) Thoneman, P. C., J. Sci. Instruments, 23, 217 (1946). (324) Timmis, L. B., J.SOC.Chem. Ind., 63,380 (1944). (325) Trans. Am. Inst. Mining Met. Engrs., 162,369-412 (1945). (326) Tsiklis, D. S., Zavodskaya Lab., 12, 632 (1946). (327) Turkel’taub, A. M., and Fainberg, M. M., Ibid., 11, 899 (1945). (328) Turner, N. C., PetroZeum Refiner, 22, 140 (1943) : U. S. Patents 2,398,817-18 (1946). (329) Twigg, G. H., Trans. Faraday Soc., 42, 657 (1946). (330) Uhrig, K., Roberts, F. M., and Levin, H., ISD. ENG.CHEM., ANALED., 17, 31 (1945). (331) Utter, N., and Kling, R., Compt. rend., 227, 41 (1948). (332) Vandaveer, F. E., Gas, 18, No. 10, 24 (1942). (333) Vandoni, R., M i m . services chim. &at (Paris),30, 18 (1943). (334) Van Slyke, D. D., Hiller, A., Weisiger, J. R., and Crus, W. O., J . Biol. Chem., 166, 121 (1946). (335) Veldheer, P. A.. Chem. Weekblad, 44, 499 (1948). (336) Vint, W. D., Metallurgia, 35, 153, 255, 294 (1946-47); 36, 47, 157, 276, 333 (1947); 37, 150, 317 (1948). (337) Voiret, E. G., and Bonaim6, A. L., Ann. chim. anal., 26, 11 (1944).

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Vorokhobin, I. G., and Filyanskaya, E. D., Zavodskaya Lab., 14, 106 (1948).

Voronkov, M. G., Zhur. Anal. Khim., 1, 285 (1946). Wagner, Georg, “Gasanalytisches Praktikum,” Vienna, F. Deuticke, 1944. Watson, C. C., U. 5. Patent 2,456,163 (1948). Weaver, E. R., and Riley, R., ANAL.CHEM.,20, 216 (1948); J . Research Natl. Bur. Standards, 40, 169 (1948). Webb, F. J., Kay, K. K., and Nichol, W. E., J . I n d . Hyg. Tozicol., 27, 249 (1945).

Webb, G . A,, and Black, G. S., IND.ENG.CHEM.,ANAL.E D . . 16, 719 (1944).

White, D. L., and Reichardt, F. E . , Gas, 25, KO.6, 38 (1949). Whiteley, A. H., J . Biol. Chem., 174, 947 (1948). Whitnack, G. C., and Holford, C. J., ANAL.CHEM..21, 801 (1949).

Wicke, E., Angew. Chem., B19, 15 (1947). Williams, D., IND.ENG.CHEM.,ANAL.ED.,17, 295 (1945). Williams, D., Haines, G. S., and Heindel, F. D., I b i d . , 17, 289 (1945).

Wilson, H. N., and Hutchinson, W., Analyst, 72, 432 (1945). Wilson, K., Ann. Botany, 11, 123 (1947). Winkler, L. T., U.S. Patent 2,387,550 (1945). Winslow, E. H., and Liebhafsky, H. A,, IKD. ENG. CHEM.. ANAL.ED., 18, 565 (1946).

Winslow, E. H., Smith, H. M., Tanis, H. E., and Liebhafsky, H. A., ANAL.CHEM.,19, 866 (1947). Wise, W. S., Chemistry & Industry, 1948, 37. Wooten, L. A., and Guldner, W. G., IND.ENG.CHEM.,ANAL. ED., 14, 835 (1942).

Zikeev, T. A., and Shifrin, M. G., Zavodskaya Lab., 13, 1130 (1947). RECEIVED November 1, 1949.

CHARACTERIZATION OF ORGANIC COMPOUNDS ROBERT L. PECK Merck a n d Co., Znc., Rahway, N . J .

T

HE characterization of organic compounds is concerned with a number of fields of experimental study. Establishment of

purity is the primary aspect of this problem. Determination of the physical and chemical properties provides a fundamental basis for recognition and identification of the pure compounds. Detailed study of the organic chemistry of a given compound, aided by the results of physical examination, ultimately leads to elucidation of structure, stereochemical configuration, and complete characterization. It is possible a t the present time to characterize new products with confidence and with relative rapidity. This is true of complex synthetic organic compounds, but applies as well to natural products, especially those which are difficult or impossible to obtain in crystalline form. Sumerous vitamins, hormones, antibiotics, and other naturally occurring compounds have been isolated, characterized, synthesized, and put t o practical application with surprising efficiency in the past few years. The characterization of these natural products is a result of the practical adaptation of physical methods and the employment of chemical reactions on a microgram scale. The increasing availability of equipment suitable for the precise determination of physical properties of small quantities of material is further reason for present efficiency in characterization work. This review is concerned primarily with certain recent contributions which have greatly facilitated characterization work in general. I t has been necessary to restrict its scope to those techniqueb and reactions which are most useful for examination of ne% compounds a t the present time. The primary problem facing the chemist who deals with very small amounts of a new substance of natural origin is the establishment, for reference purposes, of the purity of the material. hlany compounds are known which cannot be isolated free of

solvent or recrystallized without undergoing significant chemical change. For such compounds, and for others, available amounts of which are too small for extensive recrystallization or fractional distillation, criteria of purity are required before characterizing properties may be assigned with confidence. Methods now available for establishing purity of such compounds are described in the section on purity. Once purity is established, the physical properties of a pure sample of the new compound are determined. A melting or boiling point determination is made, if practicable. Physical examination, in addition to the melting or boiling point, may now include measurement of ultraviolet and infrared absorption, x-ray studies, potentiometric titration, and reduction or oxidation a t the dropping mercury electrode. The most useful physical methods now available are discussed in the section on physical properties. With comptetion of physical examination of pure organic compounds, chemical reactions provide the primary information of structural significance which, aided by structural deductions from physical studies, permits the accomplishment of partial or complete structural elucidation. Reactions of most general application are treated in the section on chemical reactions. PURITY

The classical procedures for preparing organic compounds in a pure state have long been crystallization to constant melting point for solids and distillation to constant boiling point for liquids. It is generally accepted (4,IS, d S , 9 2 ) that a satisfactory test of purity should include a test of identity and an attempt a t separation (14). The procedures mentioned above satisfy this requirement, but in many instances cannot be used either because sufficient material is not available, or because the compounds under investigation are unstable when subjected to the

ANALYTICAL CHEMISTRY

I22 conditions of the method. I t has become necessary to deal often with very small amounts of material, frequently amorphous in nature, which are nevertheless of such importance that establishment of purity is essential. Three methods for determinat'ion of purity have been develenantiomorph gives a detectable change in vapor pressure; the results indicate that a test of optical purity might be based on this procedure. Countercurrent Distribution. An apparatus and a process io1 conducting multiple quantitative ext,ractions in sequence, with small quantities of materials and in a relatively short space of time, have been developed by Craig (24). This technique may be applied to microgram and milligram quantities of material with excellent precision. Because it is possible to carry out exact mathematical computation of the distribution of a subst,ance possessing the partition coefficient shown by the test substance, the results may be evaluated with confidence. Det,ails of the process and apparatus have been adequately described in recent reviews (2.92,23). This method possesses inherent adva,ntages in that it permits the estimation of composition with precision, provides a precise measure of the partition coefficient of the substance in question, and gives an accurate estimate of the amount and partition coefficients of impurities if these be present. Chromatography. The use of chromatographic techniques for separation of many classes of organic compounds is now well known. The application of these techniques to testing purity of organic substances is becoming more frequent ( I S ) . Chromatography of a given substance will usually provide satisfactory evidence of purity if no separation occurs with adequately varied adsorbents and solvents. At the same time, the behavior of the substance under investigation provides a characteristic reference property for identification purposes. Thus, the rate of migration of a single organic substance through a chromatographic column is a function of the chemical nature of the substance, the nature

the adsorbent, and the specific developing solvent employed In general, the presence of extraneous dissolved substances affects the position of the desired substance on a column only if these substances are more strongly adsorbed. Less strongly adsorbed components proceed ahead of the desired compound and ordinarily do not interfere. An organic substance may usually be said to be pure if no separation occurs Then it is chromatographed on two or more adsorbents in solvents of very different polarities. Very careful evaluation of all available chromatographic data should be made, however, before reaching a final conclusion as to absolute homogeneity of sample by chromatographic evidence. Several general chromatographic procedures are available, including the orthodox, columnar chromatogram with sectional or flowing elution (69, 88, 101, 108), frontal and displacement analysis (18), and partition chromatography on silica gel (62, 63, 105), paper (21,52,87), and starch (59, 60, 61, 86). The use of ion eschange resins has provided an extremely useful tool for separations (@), and has found some adaptation to qualitative identification by chromatographic techniques ( 2 ) . A modification of the paper chromatographic process applicable to small scale preparative work is the recently introduced chromatopile technique (68). This procedure, employing a column composed of hundreds of close-packed filter paper disks, permits excellent separation of resolved samples. Quantities of material up to at least 0.5 gram may be conveniently fractionated in this way, and pure fractions recovered in amount sufficient for adequate characterizing examination. Each of these procedures has inherent advantages for certain types of work. Application of partition chromatography on starch to the separation of amino acids has given an elegant method which permits simultaneous qualitative identification and quantitative estimation of the individual amino acids (69, 60, 61, 86). This procedure supplements and extends the outstanding qualitative methods of paper strip chromatography for identification of amino acids. The adaptation of paper strip chromatography to the qualitative identification of sugars (10, 27, 73, 74), purines and pyrimidines (36, 94), penicillin (44),streptomycin (103), vitamin BI2 (104), and other organic compounds illustrates the applicability of the general method to qualitative identification work. The ready adaptability of paper strip chromatograms of both colored and colorless substances to examination by indicators, chemical reagents including oxidizing, reducing, color-forming, and precipitating agents, employment of ultraviolet absorption polarography, enzymatic treatment, and microbiological assays makes this method of investigation of organic compounds attractive from the identification standpoint. Moreover, the r e quirement of sample for qualitative characterization is small, OD the order of 5 to 100 micrograms. However, the results obtained by paper strip chromat.ography should be verified wherever possible by the more orthodox methods of organic chemistry. I t i8 anticipated that eases will be encountered where the paper strip chromatographic method will fail to supply adequate differentiation for satisfactory identification. In view of extensive reviews on chromatographic procedures (13, 69, 87, 88,101, 105) no further comment is made here. t ~ f

PHYSICAL MEISURE.MENTS

Organic compounds are ordinarily examined for melting or boiling point as a first step in characterization. The introduction of the Kofler micro melting point apparatus ( 4 6 ) has enabled accurate melting points to be obtained with quantities of sample in the microgram range, in fact, a t levels limited only by the facilities for manipulation of samples. The use of the microscope also permits much closer observation of phenomena occurring during heating of sample, such as transition points, sublimation, color changes, and loss of anisotropicity. Micro boiling points may now be readily determined ( 1 7 ) .

V O L U M E 2 2 , NO. 1, J A N U A R Y 1 9 5 0 Suinerous measurements such as specific gravity, index of re fraction, optical rotation (33) and dispersion, neutral equivalent, iodine number, and similar determinations which have long been generally available and are useful in characterization work need only be mentioned in passing. Many useful physical procedures have become generally available only in the past few years. Infrared spectrography, x-ray measurements, mass spectrography, polarography, potentiometry, Raman spectroscopy, ultraviolet absorption spectrophotometry, and improved light absorption spectroscopy have been adapted for general application. The results of examination by these procedures provide a very helpful asset for characterization of organic compounds. These are mentioned in more detail in the following sections. Spectroscopy and Spectrophotometry. Useful identification characteristics of colored organic compounds are provided by their specific absorption of radiant light energy (54, 99, 100). Equally valuable are the specific absorption spectra exhibited by organic compounds in ultraviolet radiation (81, 99, la)). Although neither of these characteristics is usually sufficient ES full identification, they are valuable as supplementary properties. Ultraviolet studies were of value in defining conditions under which penicillin underwent various structural modifications, and were helpful in characterization of rearrangement and degradation products of the penicillins (107). A clue to the presence of a nitrophenyl residue in the molecule of chloramphenicol (chloromycetin) was provided by ultraviolet absorption spectra (80). This lead facilitated the organic chemical degradation studies which resulted in early elucidation of the structure of the antibiotic. Characterization of a, @-unsaturated ketone groups by means of the strong absorption band appearing in the region of 230 to 260 p has provided a tool especially useful in sterol investigations. Raman spectroscopy is being studied to an increasing extent and appears capable of providing both identifying characteristics and structural information ( 9 ) . The application of the mass spectrograph to identification of organic compounds, especially isotopic materials, is a t present somewhat limited, but ample evidence is available that this type of observation provides a useful tool in cases such as gas analysis and will probably have increasing application within a few years (356). Infrared Absorption Measurements. Infrared absorption spectra frequently provide a more nearly absolute characterizing property of an organic compound ( 5 ) . Moreover, recognizable absorption due to groups such as S H , OH, CH, carboxylic acid, carbonyl, ester carbonyl, and monosubstituted amide carbonyl may readily be distinguished and provide very helpful guides to structure elucidation. Infrared spectra of penicillin and penicillin derivatives (91) furnished evidence against the oxazolone formula tentatively assigned to penicillin. The strong organic chemical evidence for the p-lactam structure provided by hydrogenolysis studies ( 4 2 ) was supported also by infrared studies of the compounds in question. I n the elucidation of structure of chloramphenicol (go), the organic chemical structure work was also aided by infrared spectrum studies. X-Ray Measurements. X-ray diffraction patterns of crystalline organic substances provide a very powerful means of identification (29, 45). The determination of unit cell dimensions furnishes a guide to molecular size and shape and may be used as a method for determination of molecular weight. Unit cell measurements provided valuable information in the structure work on penicillin (19, 25). The extent to which x-ray studies may be applied to organic chemical structural elucidation is well indicated in the work on penicillin (25). Confirmation of molecular weight and indica+ions of flat molecular configuration were given for biotin by x-ray measurements (28). Polarography. The polarngraph has been successfully applied

123 to identification and quantitative estimation of a wide variety of redox groups (67, 97). With pure compounds posswsing an oxidizable or reducible group of known type, polarography yields information on equivalent weight, as well as usually satisfactory identification of the nature of the group. Potentiometry. The improvement in techniques of potentiometric titrations of milligram to microgram amounts of sample has extended the availability of a most useful procedure (56) Potentiometric studies of hydrolytic cleavages provide highly valuable information on the products with respect to compound types and acidic and basic strength, as well as information on rates of hydrolysis. Potentiometric investigation of redox and oxidative reactions also may be carried out on very small scale samples with useful results. Equivalent weight data are often most easily determined by potentiometric measurements. Dissociation constants determined by potentiometry provided valuable information in the characterization of penicillin and ite derivatives (107). Molecular Weight Determination. Determination of molecular weight by various means has long been possible. Adaptation of various methods to the milligram and microgram scale in recent years has supplemented other phases of characterization of complex organic substances. The Northrop diffusion method ( 7 1 ) , the Rast procedure ( 7 9 ) and its modifications (70, 7 6 ) , and the Barger isothermic distillation procedure (3)and its modificatione (57, 7 0 ) all represent useful techniques which have been adapted to a conveniently small scale. The ebulliometric (89, 90) and cryoscopic (83)methods adapted to a micro basis provide accurate procedures which are very frequently applicable. CHEMICAL REACTIONS

Subsequent to Pregl’s introduction of techniques for quantitative organic microchemical analysis (78), there have been everincreasing development and extension of methods of elemental analysis and group analysis (31, 84, 102). The results of such studies have provided a sound basis for analytical characterization of organic compounds. Bccurate, significant data may readily be had on elementary composition, empirical formulas, equivalent and molecular weights, and the equivalent proportions of such groups as hydroxyl, amino, 0- and N-acyl, alkoxyl, alkimino, C-methyl, carbonyl, carboxyl, and halogen groups (17, 49, 78, 82). Methods for micromanipulation of small-scale organic reactions have generally been developed as the occasion demanded; these techniques have been extensively discussed in recent reviews (6, 7 , 102). The presence of a wide variety of characteristic organic functional groups may be detected by application of specific, qualitative tests worked out for microgram quantities using spot test techniques (30). Knowledge of the nature of functional groups present in a molecule of incompletely known structure may enable a colorimetric assay to be devised for following the color-producing moiety during degradative studies. The application of the Sakaguchi test to follow changes in the guanido groups and of the Pauly test to show concomitant formation of imidazole g r o u p in degradation of streptomycin and streptidine (18) serves as an illustration of this principle. Knowledge of functional groups also permits introduction of a wide variety of tracer groups-e.g., halogen-containing acyl groups, sulfur-containing residues, isotopic residues, radioactive groups, ultraviolet-absorbing groups, and the like-which are often of utmost importance in recognition of one or another of the fragments of complex or conjugated molecules during the subsequent degradative reactions, and may also be of value in determination of equivalent and molecular weights. More general application of such techniques is possible with the increasing availability of sensitive methods of evaluation. Microbiological assays provide a means for identification of various organic compounds possessing biological activity (86) Supporting evidence is usually necessary for confirmation of

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identity, but the remarkable specificity of microbiological assays for certain amino acids (68), vitamins (41), antibiotics (96), and various other compounds (77, 86) provides a rapid and useful tool for identification of many organic compounds as well as for quantitative estimation when they are shown to be present in a mixture. These methods are frequently helpful when mild degradation of a biologically active molecule is to be carried out. General procedures for classifying organic Compounds by solubilities and functional group reactions (I?', 278,43, 55,82) provide leads for selection of derivatives suitable for further characterization. The more commonly employed and most generally useful derivatives are fully discussed in the references cited. The value of derivatives from which t,he parent compound may be regenerated by mild conditions such as hydrogenolysis--e.g., carbobenzoxy derivatives (8, 16, 16)-is emphasized. Such derivatives are of especial import,ance when it is necessary to regenerate a derivative of a biologically active substance to recover original active substance. The choice of Iris commonly used derivatives will usually be determined by coilsiderations of specific need, such as ease of recrystallization or regeneration and availability of known or obtainable reference compounds. In so far as kn0n.n and previously characterized organics oonipounds are concerned, the general chemical methods cited above, together with the physical methods of examination discussed in the preceding sections, usually provide more than adequate means of differentiation from other compounds, of identification, and of recognition of organic structure. New and previously unknown compounds when examined by these methods will yield physical and chemical properties sufficient for recognition and identifieation purposes. When, however, it, becomes necessary to elucidate the full organic chemical structure and stereoisomeric configuration if this is involved, an extensive and detailed study of the behavior of the new molecule in a wide variety of specific chemical reactions is usually required. Comparison of suitable derivatives or degradation products or both with known reference compounds must be carried out. The selection of reactions will be based on the leads gained from preliminary observations which will have giveii considerable information on the probable size, type, and empirical formula of the molecule in hand. For example, it is ordinarily possible to determine with reasonable certainty whether hydroxyl, amino, alkoxyl, acyl, carbonyl, or carboxyl groups, unsaturated bonds, aromatic rings, sulfur, phosphorus, or halogens are present. Discussion of reactions found particularly useful for degradative characterization work is limited in t,his review to a few general types. The most useful reactions for degradative purposes are obviously those which are applicable to the milligram to microgram scale, which give quantitative or semiquantitative recoveries of product, or which provide quantitative information on the extent of oxidation, hydrogenation, or other desired reaction, and which are conclusive in terms of structural significance. Hydrolysis, acid or alkaline cleavage of conjugated molecules, is a fundamental reaction from which the fragments may be isolated for further study. Potentiometric examination of the reaction during its course often yields helpful information on basic strength of formed products, dissociation constants, an'd rate of reaction. The use of physical measurements-e.g. , potentiometry, ultraviolet absorption, infrared absorption, optical rotation, and the like--for following cleavage reactions, procedures which are now possible as a result of increased availability of suitable instruments, has augmented the accepted value of hydrolytic reactions. The recent work on elucidation of structure of actadione (47) is of interest in this connection. The degradative work on penicillin and its derivatives provides many illust,rations of t,he structural significance of such studies (20, 107). Hydrogenation reactions have become increasingly important in characterizing work, for the yields of product are usually high, t,he reactions generally go to completion, and the resulting prod-

ANALYTICAL CHEMISTRY ucts posbesv properties favorable for subsequent treatment. The recent developments leading to wider application of hydrogenation procedures justify some discussion of these reactions. Quantitative hydrogenation in neutral solution with platinum ratalyst and hydrogen a t atmospheric pressure effects reduction of aliphatic carbon to carbon double bonds, unhindered carbonyl groups, nitro groups, nitriles, and oxygen- or nitrogen-substituted benzene rings (1). Unsubstituted and alkylated benzene rings are resistant to these conditions, but are reduced when mineral acid is present in the medium. The quantitative determination of molar equivalents of hydrogen absorbed by an organic compound under controlled conditions provides valuable structural information. Hydrogenation of double bonds usually yields products nhich are more stable than the parent compound and may be handled more easily. Sulfur hydrogenolysis reactions with Kaney nickel catalyst (64) have proved to be of utmost importance in dealing with compounds containing sulfur in the form of sulfhydryl, sulfide, disulfide, thio ester, and thio ether groups (40, 41, 48, 61,66). The quantitative replacement of the iulfur atom by two hydrogen atoms gives a desthio derivative which is usually more stable than the sulfur-containing molecule, and rrhich can be much more readily handled in subsequent reactions. The desulfurization of biotin (98) was conclusive in establishing the correct structural formula for that compound. The extensive application of the method during penicillin studies further illustrates the utility o f the reaction (42, 63, 76). The reaction may also be employed to good advantage when aldehyde groups are to be converted to methyl groups (106). Formation of the diethyl mercaptal derivative followed by desulfurization with Raney nickel catalyst yields the desired methyl group. This method was employed in formation of tetraacetylbisdesoxystreptobiosamine (48) during studies on streptomycin structure. The use of Raney nickel catalyst, in sufficiently large quantity relative to sample, effects hydrogenation of carbon to carbon double bonds, carbonyl, azoxy, and hydrazo linkages and the like without the presence of added gaseous hydrogen (65). The hydrogen involved is that adsorbed on the catalyst. Nonactivated benzene rings, aliphatic acids, and esters are stable to reduction under these conditions. -4romatic alcohols may thus be converted to the corresponding aromatic hydrocarbons, as may aromatic aldehydes; thus a t 78' benzaldehyde gives toluene and not benzyl alcohol, whereas a t 25 O benzyl alcohol is obtained The temperatures required for these hydrogenations are reasonably low, ranging from about 25' to 100" C. Hydrogenation of carbon to carbon double bonds in compounds containing divalent sulfur using hydrogen and palladium on carbon or palladium on barium sulfate has been worked out for use in the generally available l o a pressure (below 50 pounds) hydrogenation apparatus (64). Among numerous oxidative reactions, which have been employed in degradation of organic compounds, the reaction of periodic acid with glycols (60, 61) is of outstanding importance. The reaction is subject to quantitative interpretation, may be used with very small amounts of sample, and yields highly significant structural information. Media which have been employed for this oxidation include acid, neutral or buffered aqueous solutions, and aqueous-organic solvent mixtures. I n compounds containing two adjacent hydroxymethylene or primary aniinomethylene groups, there occurs oxidative cleavage of the carbon to carbon bond joining them, with formation of two aldehyde groups. If more than two adjacent hydroxymethylene or aminomethylene groups are present in the molecule, the carbon moiety of each of those lying between the end members of the series is oxidized to formic acid, which may be determined quantitatively. Terminal hydroxymethylene groups are converted to formaldehyde by the oxidation, in which case quantitative estimation by dimedone precipitation permits accurate deduction as to the number of such terminal groups in the molecule. An elegant elucidation of

V O L U M E 2 2 , NO. 1, J A N U A R Y 1 9 5 0 st.ructure of streptamirie depended primarily upon periodate oxidation reactions ( 1 1 ) . Application to determination of structure of chloramphenicol (80) further illust’rates the utility of periodate oxidation. -4n extensive review adequately covering details of the methods of periodate oxidation and applications to structural work on sugars and other substances has recently appeared (39). It is beyond the scope of the present review to present more than a brief survey of the more significant recent developments in characterization of organic compounds. The wider application of sensitive physical met,hods has greatly facilitated organic structural work, although fundamental organic chemical reactions still provide the basis for ultimate elucidation of structure. However, the coordination of both chemical and physical observations is necessary for most efficient charact,erization. ACKNOWLEDGMENT

The author wishes to acknowledge t,he assistance of Paul Gale in searching the liternture. LITERATURE CITED

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Jeanloz, K., Prins, D. A , . and Reichstein, T., Hrlc (’him. Acta, 29, 371 (1946). Johnson, B. C., “Methods of Vitamin Determination.” Minneapolis, Minn., Burgess Publishing Co.. 1948. Kaczka. E., and Folkers, K.,“The Chemistry of Penicillin,” Chap. IX, Princeton, N. J., Princeton University Press, 1949. Kamm, O., “Qualitative Organir Analysis,” 2nd ed., New York. John Wley & Sons, 1932. Karnorsky, M.L., and Johnson, M . J., A s i I , . CHEM.,21, 1125 (1949). Kaufman, H. S.,and Faukuchen, I., Ibid.,21, 24 (1949). Kofler. 8 . . “Micromethoden zur Kennaeichnune oreanivcher Stoffe und Stoffgemische,” Pliiladelyhia, Arthur HrThomas Co., 1947; leading references. Kornfeld, E. C., Jones, R. G., and Parke. T. V., J . A m . (‘hem. S O C . , 71, 150 (1949). Knehl. F. A., Flvnn, E. H.. Brink, N. 0.. arid Folkers. K.. Ihid., 68, 2096-( 1946). Kunin, R., ANAL.CHEM.,21, 87 (1949). Malaprade, L., Bull. soc. chim., 43, 683 (1928). Malaprade, L., Compt. rend., 186, 382 (1928). Martin, -4.J. P., A n n . N . Y . Acad. Sci., 49, 249 (1948). Martin, A. J. P., and Synge, R. L. M., Biochem. J . , 35, 91. 1358 (1941). -Mellon, M. G., ANAL.CHEM.,21, 3 (1949). Meyer, H., ”Analyse und Konstitutionsermittlung organischer Verbindungen,” 6th ed., W e n , J. Springer, 1938. Michaelis, L., “Physical Methods of Organic Chemistry,” Vol. 11, p. 1051, New York, Interscience Publishers, 1946. Miller, G. A,, Science, 92, 225 (1940). Mitchell, H. K., and Haskins, F. A., Ibid., 110, 278 (1949). Moore, S., and Stein, W. H., A n n . 9.Y . Acad. Sci., 49, 285 (1948). Moore, S.,and Stein, W. H., J . Biol. Chem., 176, 367 (19481 Ibid., 178, 53, 79 (1949). Mozingo, R., U. S.Patent 2,457,392 (1948). Mozingo, R., and Folkers, K., “The Chemistry of Penicillin,” Chap. XVII, Princeton, N. J., Princeton University Press. 1949. Mozingo, R., Harris, S. A,, Wolf, D. E., Hoffhine, C . E.. Jr., Easton, N. R., and Folkers, K., J . A m . Chem. Soc., 67, 2092 (1945). Moaingo, R., Spencer, C., and Folkers, K., Ibid., 66, 1859 (1944). Mozingo, R., Wolf, D. E., Harris. 8 . A , , and Folkers, K., Ihid.. 65, 1013 (1943). Muller, 0. H., “Physical Methods of Organic Chemistry,” Yo], 11. p. 1115, New York. Interscience Publishers, 1946. National Dairy Research Laboratories, Oakdale, L. I., N. Y . , “Manual of Microbiological Methods of Assaying Vitamins and Amino Acids,” 1949. New York Acad. Science, An7~..V. Y . Acad. Sci., 49, 141-326 (1948). Niederl, J. B., and Niederl, V., “Micromethods of Quantitative Organic Elementary Analysis,” pp. 166-86, Kew York, John Wiley & Sons, 1938. Northrop, J. H., and Anson, M . L., J . Gen. Physiol., 12, 543 (1929). Northrop, J. H., and Kunitz, M., Ibid., 13, 781 (1930). Partridge, S . M., Biochem. J., 42, 251 (1948). Partridge, S. M., and Westall, R. G., Ibid.,42, 238 (1948). Peck, R. L., and Folkers, K., “The Chemistry of Penicillin,” Chap. V I I , pp. 159, 165, Princeton, N. J., Princeton University Press, 1949. Pirsch, J., Ber., 65, 862 (1932). Porter, J. R., “Bacterial Chemistry and Physiology,” New York, John Wiley & Sons, 1946. Pregl, F., Ber., 44, 553 (1911); “Die Quantitative Organische Microanalyse,” Berlin and Vienna, J. Springer, 1912. Rast, K., Ber., 55, 1051, 3727 (1922). Rebstock, M. C., Crooks, H. M., Jr., Controulis, J., and Barts, 8 . R.. J . A m . Chem. Soc.. 71. 2458 (1949). Rosknbaum, E. J., ANAL.CHEM., 21, 16 (1949). (82) Shriner, R. L., and Fuson, R. C., “Systematic Identification of Organic Compounds,” 2nd ed., New York, John Riley & Sons, 1940. (83) Skau, E. L., and Wakeham, H., “Physical Methods of Organic Chemistry,” 1’01. I, p. 1, New York, Interscience Publishers, 1945. (84) Smith, W. T., and Shriner, R. L., ANAL.CHEM.,21, 167 (1949). (85) Snell, E. E., Physiol. Rev., 28, 255 (1948). (86) Stein, W. H., and Moore, S.,J. B k l . Chem., 176, 337 (1948). ( 8 7 ) Strain, H. H., - 4 ~ . 4CHEM., ~. 21, 75 (1949).

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ANALYTICAL CHEMISTRY

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(99) R.’est, IT.,“Physical Methods of Organic Chemistry,” Vol. 11. p. 737, New York, Interscience Publishers, 1946. (100) I b i d . , p. 823. (101) Williams, T. I., “Introduction to Chromatography,” London. Blackie and Son, 1946. (102) Willits, C. O., ANAL.CHEM.,21, 132 (1949). (103) Winsten, W.A., and Eigen, E., J . Am. Chem. Soc., 70, 3333 (1948). (104) Winsten, W. 8., and Eigen, E., J . Biol. Chem., 181, 109 (1949) (105) Wintersteiner, O., Boon, W. R., Carrington, H. E., MacCorquodale, D. W.,Stodola, F. H., Wachtel, J. L., Coghill, R. D., Risser, W.C., Philip, J. E., and Touster, O., “The Chemistry of Penicillin,” Chap. V, Princeton, N. J., Princeton University Press, 1949. (106) Wolfram, M. L., and Karabinos, J. V., J . Am. Chem. Soc., 66 909 (1944). (107) Woodward, R. B., Keuberger, A., and Trenner, N. R., “The Chemistry of Penicillin,” Chap. XIV, Princeton, N. J.. Princeton Gniversity Press, 1949. (108) Zechmeister, L., and Cholnoky, L., “Principles and Practice of Chromatography.” New York, John Wiley & Sons, 1942 RECEIVEDDecember 17, 1949

BIOCHEMICAL ANALYSIS PAUL L. KIRK AND E. L. DUGGAN, University

T

HE biochemical analyst is confronted with a set of problems

quite different from those of chemical analysts. Classical analytical chemistry is concerned primarily with identification and determination of the ultimate units, elements, and radicals of the chemical system; biochemical analysis is directed toward the operating or functional units of the complex biological system. The functional units may be on a cellular, high molecular, or simple molecular level. Thus the carbon content of a protein has little significance as compared with the active groups and functional units of that protein. The fat content of the biological system is far less significant than the activity and structure of lipoprotein or other lipide complexes. In a similar way, the phosphorus content is unimportant except as it indicates active metabolism through the mediation of numerous phosphate compounds, stable and unstable. Biochemical analysis is inherently a more complex and broad field than classical chemical analysis. Because the major activity of the biochemist is always to find out [‘what’’ and “how much,” biochemical analysis actually must include virtually every biochemical technique. Even in the study of rate and mechanism, analysis a t appropriate intervals and under special conditions is the primary requirement. Almost all of the specialties of the chemical analyst must be considered as tools of the biochemist also. Of necessity, any discussion of the field must overlap greatly the specialized subjects already reviewed in considerable detail in this and other journals. Further complication arises from the fact that a single biochemical problem may call for the application of widely diverse techniques, or at least for a difficult choice between a number of possible techniques. The preparative stage of most biochemical analyses consists of some type of fractionation or separation. These processes are then a most important part of the field reviewed. The further application of classical procedures for analysis of elements or radicals in the fractions usually follows as a requirement for quantitative evaluation of the fractions. This rather secondary phase has preempted much of the attention given biochemistry by the analyst. Sampling procedures in biochemistry do not aim only a t uniformity and therefore total representation of a large system; more often these procedures are concerned with the minutiae of the heterogeneous system. I t is more significant to know the variations from tissue to tissue and from part to part than to determine the average composition of the whole. Even more important,

of California, Berkeley, Calif.

any knowledge that can be obtained without alteration of the system is more valuable than information obtained after applying the destructive techniques of ordinary chemical analysis. From the biochemical standpoint, the ultimate aim is to obtain analytical data from completely unaltered biological systems. Because the ultimate unit of life is the cell, the final goal must be an analytical understanding of the component structures and composition of the cell. This dictatorship of the cell determines the units and their relative importance. Because the cell is small and contains many significant components in small amount, specific. cytological and microchemical techniques of proved sensitivity are required. The necessary sensitivities have been only partially achieved, and in very limited aspects. For these reasons, development of a biochemical analysis method is one of the most important needs for the elucidation of the mechanisms and nature of life processes. The considerations treated briefly above are kept in mind in choosing the material reviewed here. The final value of any r e search publication is considered more on the basis of its probable future value than of its past accomplishments. Therefore, emphasis is placed on those techniques for which a continuing future application can be foreseen, rather than on those which appear t,n be approaching the limit of their utility.

FRACTIONATION PROCEDURES Every analysis proceeds through two stages, the preparative and the determinative. The first stage is chosen on the basis of the nature of the sample, the interfering materials present, and t o a certain degree, the nature of the determinative step. I n biochemical analysis, this preparative step is the most important I t usually consists of a fractionation of one type or another, and may be carried out a t the simple molecular level, the high molecular level, or the cellular level. SIMPLE MOLECULAR LEVEL

Two of the more important fractionation methods a t this level are chromatography and extraction. Both may be expected to have an increasing rather than diminishing value in biochemistry. Chromatography. This highly valuable technique hm been reviewed in this journal (178, 277) and elsew-here (73, 105, 208, 328). Its most significant use has undoubtedly been in the separation and isolation of biological materials, for which reason