Characterization of Organic Compounds - Analytical Chemistry (ACS

---

Characterization of Organic Compounds ROBERT L. PECK AND PAUL H. GALE Merck & Co., Znc.,Rahway, N . J .

T

A reversed-phase partition chromatography was described b y Partridge and Chilton (62), in which powdered glass is used as carrier for a stationary, n-ater-immiscible phase. Craig, Hausmann, Ahrens, and Harfenist (18) have discussed application of nests of thin glass shells in a convenient apparatus for drying as an aid in rapid determination of total solids samples. This equipment, easily made in any laboratory, is useful for determination of weight curves in chromatographic column processes, as well as in countercurrent distributions. Extension of theoretical treatment of the chromatographic separation process is much to be desired. Mowery (56) has reported data on chromatography using adsorbents of controlled particle Fize, and working pressures up to 120 pounds per square inch in borosilicate glass columns. Some theoretical considerations are given in this report. Apparatus that d l 1 facilitate experimental work should encourage theoretical as well as experimental study. Further reference to chromatography will be found in other papers in this issue. An apparatus and procedure for separation of a wide variety of charged molecules by continuous electrophoresis and ionophoresis on filter paper, described by Durrum (24), provide a method that should prove useful for evaluating homogeneity of samples. The method represents a simplification and modification of the continuous electrophoresis procedure of Svensson and Brattsten (82), and permits small scale preparative separations to be carried out in addition to analytical separations, Additional data on application of differential vapor pressure measurements to determination of purity in a convenient modification of the solubility analysis method were reported by Hughes, Williams, and Young (58). The method appears a p plicable to a variety of compounds. The importance of caution in the interpretation of paper strip chromatograms for identification purposes (64, 66) is again indicated by observations of Carter, Hearn, and Taylor (fl), who isolated from streptothricin a substance (Compound A) isomeric with lysine. The two compounds behaved in substantially identical fashion in paper strip chromatograms, but the isomeric compound was shown to be a new diamino-C8 acid.

HE accepted requirements for full characterization of an organic compound include establishment of purity of sample, determination, of physical properties, determination of elementary composition, functional groups, and empirical formula, and elucidation of structural formula and spatial relationships. It is evident that identification of a known molecule will involve less extensive investigation than full characterization of a complex new compound. The characterization of simple or complex compounds may be accomplished by any of a Tide variety of techniques and methods, some of which are more effective than others. The choice of criteria and procedural steps is often dependent on availability of special equipment. Infrared absorption spectra and x-ray diffraction patterns alone, for example, are sufficient to identify many known substances; identification carried out by chemical methods, while sometimes more timeconsuming, is equally effective. A consequence of the recent increase in number and scope of chemical studies of naturally occurring organic substances is seen in the widely held view that thorough training in principles of characterization is of fundamental importance for students majoring in organic chemistry. A comprehensive treatise on detailed methods and techniques of characterization may some day be available. I t is probably true, however, that after successfully completing the characterization of a complex, new organic molecule the chemist enjoys a thrill of satisfaction derived in large part from the realization that the sequence of steps used was not given to him in a single reference work but was the result of his own experience, insight, and skill (and perhaps some luck). The present review of recent contributions in this realm of characterization is concerned with methods for determining purity of samples, procedures for establishing physical properties, and chemical methods of characterization. PURITY

It is essential for clear-cut determination of properties and for interpretable degradative work that the samples employed be substantially pure. Procedures which are now found most useful for establishment of degree of purity of samples were reviewed recently (64, 65). No fundamentally new methods of these types have been introduced during the past year, but there have appeared a number of papers in which extensions or new applications of method are described. The development by Craig (16, 64) of a machine for carrying out many-plate countercurrent distributions, and the recent introduction of a convenient glass apparatus for larger numbers of separation stages (19, 65) are now widely recognized. These methods have been introduced in many laboratories and have proved their worth. Continued studies by Craig and his coworkers have culminated in the development of an automatic control device for use with the glass apparatus (17). A strictly discontinuous extraction train containing 220 glass equilibration cells may now be operated automatically, so that 800 equilibration stages (about 150,000 extractions) areobtainable in about 24 hours. Using this equipment, a decision on purity of sample may be reached with a considerable degree of certainty. The high resolving power possible with this system is of great importance in cases where more orthodox separations are not feasible by reason of lack of material or instability. I t is especially valuable where the homogeneity of sensitive, biologically active principles must be determined. In chromatographic procedures for determination of purity, various modifications have been reported during the past year.

PHYSICAL PROPERTIES

Infrared spectroscopy has continued to find vide application in characterization work (64, 66). Most organic compounds yield infrared spectra which provide unique reference data for comparative purposes. Furthermore, such spectra serve to indicate the presence o r absence of many functional groups including hydroxyl, amino, keto-, aldehydo-, or ester-carbonyl, carboxyl, and monosubstituted amide. The further application of infrared spectroscopic characterization for structure elucidation is limited only by the scope of accumulated data on various organic structure types. Current work along these lines is exemplified by studies on normal and isosteroids by Josien, Fuson, and Cary (43) and on small-ring compounds by Roberts and Chambers (70). The assignment of bands to particular structural groupingse.g., cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl rings-leads to greater usefulness of infrared spectra for recognition of molecule types in biological systems. Grove (34) has applied infrared in differentiating between patulin and isoclavacin, and Marion, Lavigne, and Lemay (62) found infrared of fundamental help in determining the structure of sedamine. Application of infrared in a quantitative way is further illustrated in analyses of various pharmaceutically active compounds by Parke, Ribley, Kennedy, and Hilty (61). Infrared spectra of saturated fatty acids with even numbers of C-atoms from Ce to

116

V O L U M E 2 4 , N O . 1, J A N U A R Y 1 9 5 2 C18 were given by O'Connor, Field, and Singleton (58). Perry

(67) has described analysis of mixtures of five C ~aromatic O compounds by infrared analysis TT-ith a n error of 0.5%. Analysis of solids with the mass spectrometer has been considered by Gorman, Jones, and Hipple (33). Application of this instrument to determination of nitrogen in organic compounds by Frederickson and Smith (28) has given a precision of &2%. Rock (72) has used the mass spectrograph for identification of components of mixtures of organic compounds and has stressed the possibilities of the method. The microwave spectrograph has been used by Southern, hlorgan, Keilholtz, and Smith (80) in the isotopic determination of nitrogen and carbon. Only about 0.00015 mole of gaseous product (ammonia, cyanogen chloride) is required, most of this can be recovered. T h e technique is described in detail, and mention is made of many possible applications for analysis of molecules containing other stable isotopes which can be converted to noncreative gaseous forms. The contribution of x-ray analysis to solution of problems of molecular configuration of sterols, bile acids, cycloparaffins, and analogous compounds has been discussed by Giacomello (30). The use of x-ray diffraction analysis in the identification of solid, crystalline aromatic hydrocarbons is revien ed by Hofer and PeebIes (37). The investigation of x-ray diffraction behavior of noctadecenoic acids reported by Lutton, Huber, Mabis, and Stewart (49) provides further illustration of current applications. Tabulation of data for Raman spectra of hydrocarbons and oxygenated compounds has been given by Braun, Spconer, and Fenske ( 8 ) . A small scale apparatus for determination of Raman spectra has been described by Mitsushima, Shimanouchi, and Sugita (65). Increase of sensitivity has been studied by Vacher (88). Ultraviolet absorption spectroscopy continues to be of value in structural elucidation work. Applications in lignin chemistry are noted in papers by Pew (68) and Ritter (69), who employ such data for recognition of specific phenolic moieties. Employment of ultraviolet absorption spectra for recognition of acetylenic moieties has been found helpful by Jones (42)in work carried out with acetylene compounds in his laboratory. The abTorption maxima of the acetylenic residues are so characteristic t h a t Jones was led to suggest the presence of such moieties in agrocybin and related antibacterial substances after seeing the ultraviolet spectra of these compounds (42). Ultraviolet absorption of amino acids and proteins and application to analytical investigations have been discussed by Dannenberg (20). Other recent applications have been reviened by DBrib6rB (21). The measurement of refractive index using as little as 0.25 cu. mm. of a liquid is carried out by Blohm (4)with the Abbe-type refractometer by limiting free space by means of tinfoil or aluminum foil and absorbing tissue. Precise melting point microdeterminations combined with determination of refraction of the melts has been used by Brandstatter (7) as a simple means of identifying barbjturic acid derivatives. These compounds cannot be readily distinguished by their crystal forms because of poly- and isomorphisms. Microscopic fusion analysis has been applied to sterols by Gilpin (32). Since minute quantities of sterols can be easily and rapidly recognized by this procedure, the method should be useful in the sterol field. Accurate titration of both strong and neak organic bases in various nonaqueous solvents such as benzene, chlorobenzene, nitrobenzene, chloroform, ether, or petroleum ether has been discussed by Fritz (29). Perchloric acid in acetic acid was employed and both indicator and glass electrode end points were obtained. Titrations in nonaqueous media have also been studied b l Tomicek, Blazek, and Roubal (85). Determination of primary and secondary amines in the presence of each other by treatment of the mixture n i t h acetic anhydride o r salicylaldehyde prior to titration with acid was reported by Siggia, Hanna, and Kervenski (76). Ethylene glycol-isopropyl alcohol is used as solvent Differential

117

percupiimetric titrations of amino acids and proteins permit certain deductions to be made as to structure, according to Beck ( 3 ) . Microtitration of organic acids using less than 1 mg. of sample was described by Ingold (39). The determination of molecular weight of organic compounds in organic solvents using samples down to 5 mg. in weight with precision equivalent t o that of the ebullioscopic procedure has been described by Taylor and Hall (83). The solvents do not require rigorous purification. Utilization of the degree of vapor pressure lowering in the determination of molecular weight was reported by Klages and Mohler (45). ilpplication of lightscattering studies to molecular weight determination for lactoglobulin, ovalbumin, serum albumin, and lysozyme has been described by Halwer, Nutting, and Brice (55). Determination of molecular weight of peptides by a method of partial substitution-e.g., Tyith dinitrophenyl groups-and countercurrent distribution of the partially substituted mixture has been worked out by Craig (15). Because the liberation of acidic or basic groups in peptides or reactions involving substitution of the functional groups of these substances cause striking shifts in partition ratio, countercurrent distribution permits determination of the number of such groups and ultimately the molecular weight of the parent peptide. Velick and Udenfriend (89) have presented in detail their methods of isotope derivative analysis for a number of amino acids. Positive identification is described for proline, valine, methionine, and phenylalanine a t the gamma level as p-iodophenylsulfonyl (pipsyl) derivatives from protein hydrolyzatee. Udenfriend and Velick (87) have also given details of the isotope derivative method as applied to determination of amino acid and groups in proteins. Application of the stable isotope dilution method to determination of nicotinic acid by Trenner, \Talker, Arison, and Trumbauer (86) provides an absolute assay for nicotinic acid in complex mixtures. This work is a further illustration of the importance of the isotope dilution principle in the development of specific (absolute) assay methods. A review of micromethods of determination of physical constants has been presented by Sobotka (79). CHEMICAL PROCEDURES

Quantitative aspects of functional group determination in characterization of organic compounds have been discussed by Siggia (75). Determination of functional equivalent weight by functional group analysis, verification of suspected identity, and other applications are considered. A useful summary of methods for detection, characterization, and estimation of functionat groups has been given by Viebel(92). The estimations are generally based on a titrimetric method. These procedures appear t o be representative of those comprising a practical course io identification of organic compounds for graduate students in the Technical University, Copenhagen, Denmark. Indication of a trend in quantitative functional group determinations is seen in the microanalytical use of lithium hydride by Schoniger (74). A qualitative color test for aromatic nitro and nitroso groups using lithium aluminum hydride followed by addition of aqueous sulfuric acid has been developed by Gilman and Goreau (31). The colors produced range from brown through yellow, orange, and red. Kelson and Laskowski (57) have also used lithium aluminum hydride as a qualitative test reagent for aromatic nitro groups. Another color test for nitro groups employing a sodium nitroprusside reagent or sodium pentacyanoaminoferroate after reduction was described by Okuma (59). The colors obtained may be purple, green, or blue. A color reaction for methyl ketones was reported by Adachi (2). The substance is treated with hypochlorite or hypobromite and pyridine and heated t o boiling. Compounds giving positive iodoform tests show a pink or carmine color in this test. Sakaguchi (72) has described a modification of the colorimetric determination of arginine.

ANALYTICAL CHEMISTRY

118

Organic acids may be converted to p-bromo- and p-phenylphenacyl esters in good yield by means of p-bromo- and p-phenyl-ediazoacetophenones, according t o Erickson, Dechary, and Kesling (26). Cupric chloride is used as a catalj-st. No lachrymatory products are involved, in contrast to the case with phenacyl bromides. Characterization of esters of aromatic acids and alip-aminomethyl)-morphatic dicarboxylic acids by means of S-( pholine was found by Bost and Mullen (5) to give good yields of pure, crystalline, stable derivatives whose melting point spread was wid? enough to ensure complete identification. The tertiary nitrogen atom present enables the molecule to undergo quaternization vith ease, thus affording a second derivative of the ester in doubtful cases. Bost, Starnes, and Wood (6) reported the use of 2,4-dinitrophenol as a reagent for characterizing a vide variety of halo compounds including alkyl halides, halohydrins, halonitriles, haloesters, haloketones, and haloethers. Crystalline products are easily obtainable. Microgram-scale identification of triphosphoric acid esters of thiamine by employment of quinine molybdate as a reagent and by examination under Wood's light has been described by 1-elluz and Pesez (90). The method could probably be used for differentiation of analogous compounds. Alkaline fusion reactions and alkaline degradations have been very useful in structural elucidation work for a long time. A few recent examples are given. Alkali fusions of terramycin and aureomycin yielded salicylic acid and 5-chlorosalicylic arid, respectively, in structural studies on these antibiotics conducted by Kuhn and Dury ( 4 7 ) . More detailed alkaline degradative studies on terramycin have been carried out by Pasternack, Bavley, Hess, and Conover (6s)and h y Hochstein and Pasternack (36). Alkaline degradation was employed in structural studies o n tiumulone (I,R = -OH) and lupulone [I, R = -CH,-CH =('(CH,),] by various workers including Wieland (9S), Kieland

0

O=C

&-OH

\ /

I and Mart2 ( 9 4 ) , Wollmer (96), Cook and Harris ( I S ) , and Verzele and Govaert (91). T h e value of alkaline degradation is diminished in a case such as this by reason of the possibilityof double bond migrations. Verzele and Govaert established the cited structures of humulone and lupulone by employing ozonolysis (91). Carson (10) has recently confirmed these observations in his ozonization studies. Good recovery of acetone and of methyl npropyl ketone from the volatile products as accomplished via chromatography of the 2,4-dinitrophenylhydrazones on silicic acid. The apparently specific action of osmium tetroxide in combining with steroids possessing an a,@-unsaturated 3-ketone group has been suggested by Manaro and Zygmuntowicz (61) as of potential value in detection and characterization of steroids in extracts of biological fluids. Recent advances in characterization of steroids in general are not included in the present paper. Stepwise degradation of peptides, elucidation of molecular &eights of peptides and smaller peptide fragments from hydrolyses, determination of amino acid sequence, identification of amino acid residues, and characterization of the free functional groupings present in peptides have been extensively studied. Application of dinitrofluorobenzene ( D S F B ) as a labeling reagent to introduce the dinitrophenyl (DXP) residue into peptides, and utilization of repetitive countercurrent distributions of the dinitrophenyl peptides have been employed by Craig (15) to determine molecular weights. Partial hydrolyses have been

followed by ion exchange chromatography, charcoal adsorption, and paper chromatography in methods rep( rted by Sanger (73). Enzymatic hydrolysis could be succesfully studied by these methods. Destruction of dinitrophenyl amino acids 11hen refluxed in solution in the presence of tryptophan TTW noted by Thompson (84). I n the absence of trlptophan no destruction n a s observed. Regeneration of short- or long-chain amino acids from their dinitrophenyl derivatives may be accomplished by heating the latter with ammonia in sealed tubes a t 100" for 2 hours, according to Lo\\ ther (48). T h e resulting solution is acidified and extracted n i t h ether, leaving an aqueous solution of the amino acids. The method is useful in conjunction i+ith paper strip chromatography. Further evperiments with phenylthiocarbamyl derivatives of peptides in step4 ise degradation studies have been carried out by Edmann (25). Stepwise degradation of peptides by treatment uith ethyl methyl xanthate to give an S-thiocarbethoxy derivative, folloned by removal of the terniinal amino acid as the thiazolid-2,5-dione, has been described by Khorana (44). The terminal amino acid can he recovered from the thiocarbamic acid after treatment with hydrochloric acid. The S-thiocarbethoxy products examined 11ere usually crystalline, and yields were reported to be gcod. Quantitative reaction of a peptide TTith carbon disulfide has been used by Cook and Levy ( 1 4 ) for peptide degradation on a micro scale. By means of a self-recording "pHstat," and kinetic analysis of the data thereby obtained, the number of free a-SHZ, s-NH2, or-XH