tanium complex with the reagent is unstable. When titanium was present, the thorium fluoride had a bluish tint and turned brown on ignition, because of the presence of a trace of uranium which is not carried down in the absence of titanium. Tungsten tended to precipitate as tungstic acid shortly after the sample was dissolved. An excess of hydrogen peroxide kept a limited amount of tungsten in solution but was inadequate for 20 mg. or more. If the precipitate of tungstic acid was allowed to digest for a time, it did not always redissolve when hydrofluoric acid was added, but remained with the thorium fluoride, 11 here it interfered with the titration. A set of six determinations was made in 11 hich the tungstic acid was allon ed to digest, then removed by filtration prior to the precipitation of the thorium fluoride. In this case, however, the recovery of the thorium was only about 75%. dpparently the other 25’% was carried down by the tungstic acid. When such amounts of tungsten \\ere present, best results were obtained by adding the hydrofluoric acid as soon as the alloy was in solution and neutralizing part of the acid later by adding a calculated amount of ammonia. This
prevented any tungsten from contaminating the thorium precipitate. EXPERIMENTAL RESULTS
Determinations were carried out on solutions made up to sirnulate the alloys under consideration in which the thorium content would vary from 0.1 to 3.0y0 (Table I). In a total of 161 determinations, the average thorium found \vas 99.4% of the amount taken. The relative standard deviation for individual values n a s 0.56y0,with no significant difference in the different synthetic solutions. The simulated titanium alloy was made from solutions containing uranium, thorium, and titanium. Honever, in the case of tungsten, because the main problem was t o keep it in solution while the thorium fluoride \vas precipitated, an alloy of tungsten and uranium \vas prepared and saniples from it nere dissolved in the prez-ence of known amounts of thorium. The determination m s then made according to the second procedure given. The amount of thorium that can be determined by this method is limited only by the precision of the titration. Three milligram$ !vas the smallest
amount titrated in these esperitnents, but by using a more dilute EDTA solution and, if necessary, a photometric end point, much smaller amounts could doubtless be determined. -45 little as 0.03 mg. of thorium was recovered from complex synthetic mixtures by Grimaldi and Fairchild !3). LITERATURE CITED
( 1 ) Fritz, J. S., Ford, J. J., .%SAL. CHEJI. 25, 1640 (1953). (2) Furby, E., Btoniic Energy Research Est,ab. (Gt. Brit.) C/R 1435, hIay 1954. ( 3 ) Grimaldi, F. S., Fairchild, J. G., U. S. Geol. Survey Bull. 1006, 133 (1954). ( 4 ) Hillebrand, W .F., Lundell, G. E. F., Bright, H. .%.,Hoffman, J. I., “Applied Inorganic Analysis,” pp. 533-42, Riley, Xew k-ork, 1953. (5) Iiall, H. L., Gordon, L., ANAL. CHEM.25, 1256 (19531. (ti) Scott, W.S. “Standard Methods of, Chemical Analysis,” 5th ed., Vol. 1, pp. 196-553, Van Sostrand, Xew York, 1939. ( i )Thomason, P. F., Perry, 11. A., Byerlj-, IT. M., -4s.4~. CHEXI. 21, 1293 (1949).
RECEIVED for review January 14, 1958. dccepted May 31, 1958. Work done under the auspices of the U. S. Atomic Energy Commission.
Paper Chromatography of Streptothricin Antibiotics Differentiation and Fractionation Studies MARTIN 1. HOROWITZ’ and CARL P. SCHAFFNER Institute o f Microbiology, Rutgers, The State University, New Brunswick, N. J. ,An adaptation of circular paper chromatography has been used for the survey of a large number of poorly defined, peptide-like antibiotics related to streptothricin. Circular paper chromatography was found to be superior to linear, ascending, or descending chromatographic techniques. Most of the antibiotic preparations examined revealed multiple components after chromatographic separation. The solvent system, 1 -propanol-pyridine-acetic acid-water ( 1 5 :10 :3 :12), used in these studies was alsoeffectively applied to large-scale, cellulose powder column partition chromatographic separation of these antibiotics. Application of the preparative aspects of this chromatographic technique to the qualitative and quantitative analysis of several streptothricin complexes is now under investigation.
1616
ANALYTICAL CHEMISTRY
I
search for new antibiotics, substances are frequently found which apparently belong to the large group of ill-defined antibiotics called streptothricins. Reportedly tosic, thrse antibiotics have varied biological activities against both Gram-positive and Gram-negative bacteria, fungi, and viruses. The purpose of this study was to devise rapid paper chromatographic tnethods for the separation of the various members of the streptothricin family of antibiotics, enabling efficient characterization of unidentified, niicrobiologically related antibiotics obtained from a screening program. The streptothricins are uwally referred t o as basic, Ivater-soluble substances which are insoluble in most organic solvents other than the lower alcohols. The basicity can be attributed to the presence a3 structural units of the basic amino acids, streptolidine N THE
( d ) , [also referred to as gearnine ( 3 ) roseonine ( I I ) ] , and $.e-dianiino-ncaproic acid (6,19). The nenly described aniino sugar, 2-amino-2-deosya-D-gulose has been found in streptothricin and streptolin B (,’IS). Because most of the streptothricin antibiotics exist as complexes, it has al\vaj-s bwn desirable t o obtain clrar-cwt separations and differentiations of coniponents by some convenient paper chromatographic technicjue. This has bren tlifficult,, because with a large iiuinbrr of chromatographic solvent systems, the various streptothricins 1,shibit similar R, values, as well as a marked tendency towarti streaking and tailing. A varirty of analytical approaches Present address, Gastroenterology Re: aearrh Laboratory, The Mount Sinai Hospital, S e x l o r k , S . T.
Figure 1.
Circular paper chromatogram of roreothricins
have bern applicd to the strq'tothricin antibiotics, Streptothricin was first reported in 1940 hy Waksinan and Woodruff (20). Detailid isolation and characterization studies involving ti carbon column chromatographic trchnique were described hy Cnrtrr el al. (4). The use of carbon has also bern reported in the isolation and fractionation of antibiotic 136 ( l ) ,geomycin (21, mycothricin ( l G ) , pleocidin (G), striytin @ I ) , and streptolin (7, 14) cornplrws. Actinoruhin and lavendulin (8) w r i : isolated by chromatographic proccdurrs involving the nse of Decalso and acidwashed alumina. I n identifying streptothricin VI vith streptothricin, Swart ( 1 7 ) descrihd a countercurrent distribution method ismploying solvent systems composrd of borate and bicarbonate briffrrs, straric acid as carrier, and pcntasol. Disadvantages of this countercurrent distrilrution tcchniqrie werc the marked skewina and overlamina .. of the basic components due to shifts in partition rorffieients with changes of concentrations. Lamon, Sternhcrg, and Petrrson (3) employed ion exchange paper chromatonaphy for evaluating h m m g r n d y of streptolin preparations. They ohsrrved that culture filtrates of antibiotic 1.76 fermentations contained the same antibiotic factors which iime present in the strrptolin complex The roseothricin complex was deserihed by Sahnri (16) who reported the separation of the complex by paper chromatography employing a system consisting of 1butanol, ammonia, and aqueous p toluenesulfonic acid. Rrockmann and Musso (8) isolated geomycin, and investigated the complexity of their prep-
-
Figure 2 .
Circular poper chromotogrom of streptolins
arations by means of circular paper chromatography with a system consist ing of n-butyl alcohol-pyridine-acetic acid-water (15: 10:3: 12). hiycothricin, a newer member of the strrptothricin gronp, was isolated by Schaffner, Rangaswami, and \Taksman in 195.5 ( 1 6 ) . During thc purification stiidirs ( I S ) , the complex nature of various preparations n w determined hy using a circular paper chromatographic technique with the solvent system n-propyl alcohol-pyridineacetic acid-water (15:10:3:12). A modification of the solvent system employed by Brockmann for his geomycin complex was the substitution of n-propyl alcohol for %butyl alcohol which affordrd highrr R, values, and thus bettrr rrsolution of low mohility streptothricin Brockmann employed components. the n-hutyl alcohol system R S an analytical tool for differrntiating gromycin from strrptomycin and n e e mycin. However, the ncomycins and streptomycins may be differrntiated from thc streptothricins most rradily with a variety of solvent systems. The arratrr. hut hrretofore uncxalored. valur-of this system, and of the propanoi modification, is in the differentiations within the streptothricin group. The antibiotic separations ohtained with this tcchniqnr were so promising that an rxtcndrd study was madr to survey the streptothricin group of antibiotics. The results of this survey arc also given. It has heen possible to cmploy this paper chromatotographic solvent system for the isolation of individual components on a preparative scale, through the use of paper powder column chromatographic techniques.
REAGENTS AND APPARATUS
FILTER PAPER. Whatman No. 1, 30 cm. in diameter was used. CELLULOSEPOWDER.Whatman, ashless, standard gradc cellulose powder was used as the adsorbent for column chromatography, DEvELoPING SOLVENT. The uniphase system n-propyl alcohol-pyridineacrtic acid-mater (15: 10:3: 12) was used for both the circular paper chromatography and the cellulose powder column chromatography. Fresh solvent system was prepared evcry 24 hours. ANTiBroTIC SOLUTIONS. For most preparations, approximately 3 to 4 mg. of the antibiotic was dissolved in 0.2 ml. of distillrd water. Sohitions containing up to 20 mg. of antibiotic in 0.2 ml. of distilled water were required for the ninhydrin drtection of the crude strpptolin, mycothricin, and pleocidin preparations. Chromatography of the sulfatr salt of an antibiotic supplied as a hydrochlorid~salt was effected by dissolving the antihiotic in 0.2" sulfuric acid and applying this solution to the paper. The sulfate salts were conrertrd to the hydrochloride salts by passagr of the antibiotic through a small column of IR-45 resin in the chloride phasr. .4NTIRIoTIC PREPARATIONS. Streptothricin sulfate, Upjohn €3804; streptothricin hydrochloride, Ortho; streptcthricin VI, Rutgers lot 1; pleocidin p o d e r , Sharp and Dohnie 1257 pol; antihiotic 136, Upjohn 137NR-3; strrptolin A sulfate, Wisconsin i-D44.2; strrptolin R sulfate, Wisconsin antibiotic VIIa, XI11 PV, -244; Wisconsin PV, -237; antibiotic IXa, LVisconsin; mycothricin complex, Rutgers; niycothricin, Rutgers PI; mycothricin, Rutgcrs P3; geomycin, G o t tingrn; and roseothricins A, B, and C, Nagoya were used. VOL 30, NO. 10, OCTOBER 1958
1617
Figure 3. Circular p a p e r chromatogram of mycothricin com- Figure 4. Circular p a p e r chromatogram of streptothricin VI, plex (ion exchange product), streptothricin Vi, partially resolved and pleocidin and streptolin complexes mycothricin complex (peak 1 and peak 3). and geomycin
NINHYDRINCOLOR REAGENT. Chroma,tograms were sprayrd with a pyridine-water (1 to 1) solution containing 0.25'% ninhydrin (10). CHROMATOGRAPHY CHAMRER. A vacuum desiccator, the lid having an outer diameter of 30.2 em., served as the chromatography chamber. A Petri dish, 14.7 em. in diameter, containing 80 nil. of thr solvent system was mcchsnically supported and was located 4 cm. under the paper. The paper was supported hetwecn the upper and lower lids of the desiccator. A wick of nonabsorbent cotton, 3 mni. wide and 6 cm. long, served to conduct solvent from the Petri dish to the center of the paper. cHRO&L4TOQRAPHY COLUMN. A 59 x 5 cm. length of glass pipe jointed a t the base by a sintered-glass disk served as the column. FRACTION COLLEGTOR.A Technicon fraction collector was employed in the colnmn chromatography experiments. METHODS
Preparation of Circular Chromatagrams. A small circle, 3.1 cm. in diameter, was drawn a t the center of the filter paper. Approximately 4 p l . of the antibiotic solution was applied with a micropipet along a defined arc of the constructed circle. The width of the zone of sample application did not exceed 1.5 mm. A solution of streptothricin V i was applied routinely to all chromatograms to serve as a reference control. A thin wick of nonabsorbent cotton was inserted at the center of the circle to serve as a conduit for the solvent to rise to the paper. All chromatograms were run a t 23-4' C. The paper was placed between the upper and lower lids of the desiccator 1618
ANALYTICAL CHEMISTRY
and permitted to equilibrate with thr solvent system for 1 hour. The cotton wick was then lowered into the Petri dish containing the solvent system. The duration of the chromatographic development was 5'1, hours. At the end of this period, the solvent front was generally 11to 11.5 cm. from the center of the circle. The paper was withdrawn from the chamber, and the solvent front was marked. The chromatogram was dried in a hood. Detection of Antibiotics. For ninhydrin development, the chromatograms were sprayed with the ninhydrin reagent. The chromatograms were placed in R 90' C. oven for 5 to 10 minutes. A slightly different procedure was employed for the ninhydrin detection of antibiotic miutures containing three or more components of similar R, values in that the ninhydrin development was allow~ed to proceed for 2 hours a t room temperature. Band definition was preserved, and the diffuse ninhydrin-positive background coloration was eliminated. Sections of the paper chromatogram were cut out for bioautography and placed for 10 minutes on agar plates seeded with a Bm'llus subtilis spore suspension. The plates were incubated for 20 hours a t 37' C. The zones of inhibition were compared with the ninhydrin-positive zones developrd on the other sectors of the chromatogram. Preparation of Cellulose Chromatographic Column. Using 23-gram portions, 414gramsof dry cellnlose powder (Whatman ashless, standard grade) were packed and tamped into the glass column. The packed height (vas 52 em. Column Chromatography. The packed cellulose column was aligned with the fraction collector. A 0.5294gram sample of pleocidin hydrochlo-
ride was dissolved in 12 ml. of solvent mixture, and the resulting solution was added to the top of the adsorbent, and allowed to flow through under gravity. As the level of solution dropped to the top of the adsorbent, three 15-ml. portions of the solvent system were added in succession. An additional 40 ml. of the solvent system were added, and a 1-liter separatory funnel containing the solvent system was attached to the column to serve as a reservoir for continuous feed. By regulating the height and the orifice size of the reservoir, it was possible to maintain a flow rate of approximately 48 ml. per hour. Fractions containing 13 ml. per tube were collected and analyzed by ninhydrin spot tests and by circular chromatograms. The results of the circular paper served as a guide for pooling the appropriate fractions. The pooled column effluent was processed by extraction with an equal volume of ethyl ether, and the precipitated aqueous phase which contained the antibiotic was lyophilized. The antibiotic residue was further purified by the formation, recrystallization, and reconversion of the helianthate salt derivatives. DISCUSSION AND RESULTS
Circiilar paper chromatography with the solvent system npropyl alcoholpyridine-acetic acid-water (15: 10:3:12) was used successfully for the separation and tentative identification of streptothricin-like antibiotics. Circular paper cliromatography was vastly superior to either ascending or descending paper chromatography of the streptothricins with this solvent sys
Table I is a compilation of R, values of the antibiotic hydrochloride and sulfate salts of 14 antibioticpreparations, all of which represent streptothricintype compounds. These values represent an average of several determinations (3 to 5 ) for a particular preparation. The R, values vary approximately 0.02 unit; the R, value variations may he kept to a minimum hy control of such conditions as constant temperature, use of fresh solvent s y s terns, application of the sample as a narrow hand a t a constant distance from the center of the chromatogram, use of wicks of approximately equal dimensions, and maintenance of a constant distance between the solvent reservoir and the paper. All of the components listrd in Table I possessed antibiotic activity; a s demonstrated by bioautographic tests with a Bacillus subtilis spore suspension. Of the streptothricin-like preparations tested in this solvent system, no antibiotic component exhibited an R, value higher than that of streptothricin. The antibiotic hydrochloride salts exhibited R, values higher than those of the corresponding sulfate salts. Figures 1 to 4 reprrsent chromatograms of antibiotics grouped according to similar paper chromatographic behavior. For reference purposrs, streptcthricin VI was included in each chromatogram. Substnncrs which exhibited similar R, values in the same solvent system may or may not be identical. It is advisablc to comparr such cornponents in a number of differrnt solvent
Table I.
Circular Paper Chromatography of Streptothricin4
Minimum No. of Components
.HC1
1 2 4 1 3 2 3 3 2 4 2
0.50 0.35, 0.50 0.22, 0.35, 0.42, 0.50 0.42 0.35, 0.40, 0.50 0.24, 0.33 0.30, 0.35, 0.50 0.30, 0.33, 0.44 0.30, 0.44 0.26, 0.35, 0.42, 0.50 0.40, 0.33 (diffuee
Antibiotic Streptothricin Streptothricin VI Pleocidin complex Viomycin Antibiotic 136 Streptolin A Streptolin B Antibiotic VIIa Antibiotic IXa Mycothncin "nmnlPr IU.l.r.l.. Geomycin
...
1.
0.43 0.32, 0.43 0.20, 0.32, 0.37, 0.43 0.38 0.31, 0.43 0.20, 0.33 0.27, 0.32, 0.43 0.27, 0.31, 0.41 0.27, 0.41 0.23, 0.32, 0.37, 0.43 0.35, 0.26 (diffuse
bands) bands) Roseothricin A 2 0.30, 0.50 0.32, 0.43 Roseothricin B 1 0.31 0.27 Roseothricin C 1 0.28 0.24 (in mn.) l o center bind R, = diRtnnrQ - .. _ from _ center of appliration .- znne-of eachclitirnnce tin mni.) from renier of ~pplicatimzoiie 10 solvent from
__
D
systems. Unfortunately, additional solvent systems adequate for differentia& ing the streptothricins are not available. Substances which differed but slightly in R, values may nevertheless be differentiated visually, as is evident in Figures 1 to 4. It is therefore important that test compounds exhibiting similar R, values be chromatographed alongside one another. iMultiple spot formation has been observed previously in the chromatography of basic antibiotics (id). The observation that several pure substances such as viomycin, streptothricin, and roseothricin C gave rise to hut one band in this system tended t,o remove from consideration the possibility of multiple si>ot formation. This finding was
Figure 5. Circular p a p e r chromatogram of pleocidins obtained b y cellulose powder column chromatography 2. 3.
R, Values'
Pleocidin complex, starting moterid Streptothricin VI, reference rtondord Pleocidin I
4. 5. 6.
Pleocidin II Pleocidin 111 Pleocidin IV
strengthened by the observation that rechromatography in this system of a previously chromatographed and eluted band gave rise to but a single hand. The circular chromatographic system described has been helpful in studying the purity and identity of various streptothricin-like preparations. As an example, crude roseothricin, streptolin, pleocidin, and mycothricin complexes all possess a component which exhibits the same R, value as streptothricin in the described solvent system. It was not possible to achieve satisfactory paper chromatographic separs, tion with highly complex preparations such as crude streptolin and crude mycothricin, owing to the presence in each of a large number of similarly constituted antibiotics. In such cases the application of a prior fractionation procedure, such as carbon chroms, tography, has heen found useful. The wpropyl alcohol-pyridine-acetic acid-water system (15: 10:3:12) has been used effectively in following the charcoal Chromatographic fractionation of the mycothricin complex. In this chromatographic separation on acidwashed Darco G-60, three distinct concentration peaks of antibiotic material were observed. The first peak was obtained with water development, and consisted of two components with R, values identical to the components in streptothricin VI. A second minor concentration peak was also obtained with water development. Thismaterial consisted of three components as determined by the circular chromatographic technique. Mycothricin itself was eluted in a third peak of antibiotic concentration only with acid-ethyl alclohol development. Both of the first two elution peaks appeared to he symmetrical by the ninhydrin and antibiotic assay procedures. However, variations in specific rotation were observed among the fractions, thus indicating a nonhomogeneity of the materials within the peaks. The suspected nonhomogeneity was confirmed VOL. 30, NO. 10, OCTOBER 1958
1619
by the application of this circular paper chroniatographic technique. The paper chromatographic detection of nonhomogeneity was also made among the various peak fractions from the carbon chromatographic fractionation of the streptolin complex. Dyer ( 7 ) designated the antibiotics obtained from the five discrete concentration peaks in the column effluent as streptothricin, antibiotic IXa, antibiotic VIIa, strciptolin B, and streptolin A. The complexity of each of these antibiotic preparations is illustrated in Figure 2. It appears that the antibiotic components of Rr values (for the hydrochloride salts) 0.30, 0.33, and 0.44 recur under several discrete concentration peaks. Nycothricin and geomycin both evIiihit the same type of diffuse tailing in this solvent system, making Rr Iralue determinations difficult. These two substances, however, may be differentiated by streak dilution assay because pure mycothricin exhibits a much grrater antifungal activity than geomycin. Satisfactory separations of several streptothricin mixtures on a preparative scale \yere obtained by powdered cellulose column chromatography with the solvent system n-propyl alcohol-pyidine-acetic acid-nater (15: 10:3: 12). Table I1 shows the pattern of coniponent appearance in the colwnn effluent obtained as a result of the clirornatography of pleocidin hydrocliloride (complex) on a colunin of cclllulose powder. The order of coniponents appearing in the column effluent paralleled the sequence displayed in circular paper chromatography. Figure 5 illustrates the separations effected by cellulose ponder column chromatography of the pleocidin complex. The names pleocidin I to IT' were provisionally assigned to the individual components obtained by this procedure. Pleocidin IV, which Itas detected by ninhydrin analysis of
Table II. Chromatography of Pleocidin Hydrochloride on a Column of Cellulose Powder
Fraction Eluate Volume, hI1. 0-455 456-610 611-715
Component Pigments paper throw Pleocidin I Traces pleocidin I pleocidin I1 Pleocidin I1 Traces pleocidin I1 pleocidin I11 Pleocidiri 111 Traces of pleocidin I11 Pleocidin 11-
+
716-931 932-1022 1023-1434 1435-1889 1890-2084
+ +
effluent concentrates, could not be detected in circular papergrams of the pleocidin coniplev unless very high concentrations were used (which resulted in blurring of the major zones). The isolation on a preparative scale of individual components, which also behaved as individual components v,-hen chromatographed by the circular paper technique, argues further against the possibility of multiple spot formation. The isolated pleocidin components differ in optical rotation and in antimicrobial spectrum. Details of chemical coiistitution and antibiotic properties of these components do not, lion-ever, fall mithin the scope of this article and will be reported a t a later date. Cliaracterization of isolated components and the application of cellulose powder column chromatography to the qualitative and quantitative analysis of other streptothricin complexes is being studird. ACKNOWLEDGMENT
The authors are grateful to the following for supplying antibiotics: John R. Dyer, streptolins, Hans Brockmaim, geomycin; Yoshimasa Hirata, roseothricins: and H. Boyd Woodruff, pleocidin. The authors are grateful to
Selman A . Kaksnian for his encouragement and support. LITERATURE CITED
(1) Bohonos, E., Emerson, R. L., Khiffen, A. J.. iYash. AI. P.. DeBoer., C.., Arch. Biockeitc. 1 5 ; 215 (1947). (2) Brockmann, H., Musso, H., Chem. Ber. 87, 1779 (1954). (3) Zbid., 88, 648 (1955). (4) Carter, H. E., Clark, R. IC., Jr., Kohn, P., Rothrock, J. IT., Taylor, IT-. R., West, C. A,, Khitfield, G. B., Jackson, K. G., J . A n i . Chenk. SOC. 76, 566 (1954). (5) Carter, H. E., Hearn, \T. R., Lansford, E. RI., Jr., Page, A. C., Jr.,
Salsman. 1;. P.. Shaoiro. D.. Tavlor. \I7.R., Zbzd., 74, 3704 (1952). (6) Chartie!, J., Roberts, \I-. S., Fisher, \f , P., dntibiotrcs & Chemotherapy 2,307
(1952) (7) Dyer. J. R.. oersonal communication, 1956. (8) Kocholaty, E. L. R., Kocholaty, R., J . Biol. Cheni. 168, 757 (1947). (9) Lnrson, I,. &I., Sternberg, H., Peterson. \I-. H., J . -4m.C h e w Soc. 75, 2036 (1953). (10) Noore, S , Stein, \T. H.$ J. Bzoi. CAenz. 192, 663 (1951). (11) Sahanishi, I J . -4m.Chem. Soc. 76, 2845 (1954). (12) Peterson, 1) H., Reineke, I,. lI., Ibid., 72, 3598 (1950). (13) Rarigasn-ann, G., Schaffner, C. I' \Yaksman, S A , Antzbzotzcs CP. Cheniotherapy 6, 675 (1956). (14) Rivett, R. \I-., Peterson, \I-. H.. J . A n i . Chenz Soc 69,3006 (1947). (15) Saburi, 5 , J . .Antibzotzcs ( J a p a n ) 6B, 402 (1953). (16) Schnffner, C. P., Rangaswami. G , \Vaksman, S. 3rd Intern. Congi. Biochem. Brussels, .lug. 1-5, 1955. (171 Swart. E. .%.. J . -4ttz C'hetri. Soc. 71, 29.22 (1949). (18) Van Tamelen, E. E., Dyer, J. R.,
.
Carter, H. E., Pierce, J. I-., Daniels, E. E., Ibid., 78, 4817 (1956). (19) Van Tamelen, E. E., Smissman. E. E., Ibid., 74,3713 (1952). (20) Waksman, S. A,, Woodruff, H. B.,
Proc. Soc. Exptl. Bzol. M e d . 49, 207 (19.22). (21) Koodrnff. H. B., Foster, J. K.) Bactwiol. 52, 502 (1946).
RE[ EKED for review Kovember 30. 1957. Accepted Ala!- 2, 1958.
Paper Chromatography of 2,4-Dinitrophenylhydrazones of Aromatic Carbonyl Compounds E. SUNDT and M. WINTER Research laboratories, Firmenich & Cie, Geneva, Switzerland
T
paper chromatography of 2.4dinitrophenylhydrazones of aliphatic aldehydes and ketones has been deqcribed (1-8). The present authors found that the method of Horner and Kirmse (4) using N,N-dimethylformamide-impregnated paper with Decalin HE
~
1620
ANALYTICAL CHEMISTRY
or cyclohexane as the mobile phase, gave very satisfactory results in the paper chromatography of the 2,3-dinitrophenylhJ-drazones of aliphatic aldehydes and ketones in the C1 to C9 range, if sufficient stationary phase \vas present in the atmosphere of the jar.
b A descending method is based on a two-phase solvent system, using N,N-dimethylformamide-impregnated paper as the stationary phase and a mixture of cyclohexane-cyclohexene as the mobile phase. This method is especially suited for separating and identifying the 2,4-dinitrophenylhydrazones of aromatic carbonyl compounds.
