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 . Ani. 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 . Ani. 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. Bactwiol. 52, 502 (1946).
K.)
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.
Hon ever. the 2.1-dinitroplicnylhydrazones of aromatic conipounds either did not migrate or had too low Rf values to be separated sufficiently. Schmitt. Moriconi, and O'Connor ( 8 ) separated nitrophenylh) drazones of mono- and dicarbonyl compounds using a solvent system of dibutyl etherdimethylformamide - tetrahydrofuran. Neigh ( 7 ) developed a reversed-phase method for the s q a r a t i o n of 2,4-dinitrophen?-Iliydrazones of dicarbonyl and aromatic compounds. The methods describrd in the liteiature were either too laborious or unsuitable for the authors' purposes. By using S,-T-diniethjlforniamidt~as tlie stationnry phase on Whatman S o . 7 paper and :I mixture of cyclohexane-cyclohesene (5 to 3 v. 'v.) as the mobile phase, w r y satiGfactory results IT ('re obtained in separating the 2,4-dinitroplienylhydrazones of aromatic carbonyl compounds containing one benzene ring (Table I). The solvent atmosphere in the jar proved t o be very important in obtaining good results. This system is also suitable io1 tht' separation of the 2,~-dinitrophrnylhydrazones of Ion er aliphatic carbonyl compounds (Table 11); for these clrrivatives, hou ever, Decaliii k, in g w eial, better suited as the mobile pliav. EXPERIMENTAL
Sheets of Khatnian S o . 7 filter papw (19 X 54 cni.), cut across the running direction of the fibers, nere dipped into a 50 volunie 70solution of .Y,S-dinieth>-lformaniide in acetone, then slightly preqsed between dry sheets of filter paper, and hung in the air for a diort tinie to evaporate the surplus acetone. By means of a micropipet. 3 to 20 pl. of a solution containing 1 nig. per nil. of 2,4-dinitrophenylhydrazone dissolvd in chloroform were placed on the starting line, 12 cin. from one end of 3 sheet prepared as described above. (The chloroform solutions of the derivatives must be freshly made; if not, some derivatives have a tendency to produce less round spots, or even streaks.) The prepared paper sheet was subsequently equilibrated for about 12 hour.; before the mobile phase was filled into the trough. For successful result.', the atmosphere in the chamber must be saturated with respect to the stationary phace, S,S-dimeth~lfor~iiazmide. as n ell as to the mobile phaqe, cyrlohexsnecyrlohexene ( 5 to 3 v./v.). Oncc the correct equilibrium of the tu o phase-: is obtained. tlie chamber can be run for months with very reproducible rewlts. In a chamber about 40 em. long, 30 cni. nide, and 5.5 cni. high, the t n o side ualls (30 X 55 cni.) n-erc l i n d with filter papers which n-ere partially imnierqed in crystallizing dishes containing the mobile phase, cyclohesanecyclohexene (previously saturated n ith S,S-dimethylformamide). The back wall was lined with a filter paper pnrtially immersed in a crystallizing dish
Table
I.
R, Values of 2,4-Dinitrophenylhydrazones of Some Aromatic Compounds
Ri Acetovanillone p-Hy droxybenzaldehy de p-hydroxy acetophenone
Salicylic aldehyde -4cetoveratrone .inisaldehyde Cinnamaldehy de Benzaldehyde -icetophenone p-Methylphenouyacetic aldehyde p-Propioanisolaldeh yde 3-Phenylpropionic aldehyde 1-Phenyl-butan-3-one Cuminaldehyde AIenthone 2.4-Dinitrophenylhydrazine
containing .V,S-diniethylformamide (previously saturated with the mobile phase). In addition to these three lining papers, t n o crystallizing dishes containing S,N-dimethylformaniide saturated viith the cyclohexane-cyclohexene niixture nere placed on the bottom of the chamber. After 24 hours' equilibration a t 25' C., the chamber n-as ready for use. To assure a n-ell equilibrated atmosphere, it \I as found helpful to moiqten the t n o lining papers on the side nalls every time a new chroniatograin \\asplaced in the chamber. During a development period of 3.5 to 4 hours, the mobile phase moved about 35 em. Subsequently. the chromatogram n a s dried at 50" C. The yellow spots of the 2,4-dinitrophenylhydrazones \\-ere viqible as such, but the usual qpraying with aqueous lOY0 sodium hydroxide solution increased the sensitivity; moreover, the identification of individual compounds was easier because of the vel1 known different colors, varying from yellor to hronn, red, and blue, typical of the derivatives of aromatic and dicarbonyl compounds. The examination of the chromatogram in ultraviolet light of \mve length mainly in the region of 3655 A. (Philips mercury vapor lanip, H P W 125W) gave still greater scnsitivity. I n ultraviolet light the qpots are revealed as dark shadows and even traces are detectable in this way; 0.1 y was definitely visible. RESULTS
The R, values should not be taken as abwlute. In a two-phase system as used here, the R, values always vary I\ ith the quantity of the stationary phase fixed on the paper. I n spite of a rigidly standardized procedure in inipregnating the paper sheets. differences are unavoidable. I n addition, the humidity of the air has a great influence if the chromatograms are not prepared in a room with constant humidity. The test paper chromatograms, the R j values for TT hich are stated in Tables I and 11, were prepared a t an average relative air humidity of 38y0,varying between the limits of 30 and 48%. Be-
0.05 0.05 0.07 0.15 0.18 0.23 0.34 0.35 0.42 0.43 0.45 0.47 0.54 0.64 0.84 0.05,O .32, 0.71
Color with 10% NaOH Dark brown Brown Brown Dark brown-violet Yellow-brown Brown Light brown Light brown yellow Light brown Orange-light broxn Brown Yellow Yellow Yellow
Table ll. R, Values of 2,4-Dinitrophenylhydrazones of Some Aliphatic Carbonyl Compounds
Rl Glycolaldehyde 0.06 Formaldehyde 0.29 ilcetaldehyde 0.36 Diacetyl-( mono). 0 38 -4cetone 0.44 Hex-2-en-1-a1 0.61 I-Hexanal 0.67 a Spot of this derivative becomes strongly red on spraying with aqueous 10yo sodium hydroxide solution.
cause of the variations in the Rfvaluc
a test niisture should always run 3 niultaneously on the sheet, together n ith the substances to be analyzed. The R, values increase in a logical order, depending on the decreasing polarity of the compounds. I n agreement n-ith this, p-hydroxybenzaldehyde and salicylic aldehyde are ea+ separated. The hydro\) 1 group in the ortho position to the aldehyde group remarkably decreases the polarity of salicylic aldehyde (H-bonding). Cinnama1dehS.de has, in spite of t n o more carbon atoms in the side chain, the same R, value as benzaldehyde. The conjugated double bond in the sitle chain counterbalances exactly and thcrcxfore cancels the influence of the increase in the nuniber of carbon atoms. Thus, benzaldehyde and cinnamaldehyde cannot be separated in the solvent sjsteni described here. Tahlc I shons a satisfactory separation ~f 2,4-dinitrophenylhydrazoncsof aroniatic carbonyl compounds-man) of nhich are found in natural products. The R, values obtained by running test mixtures were the same as those stated for compounds run singly. A difference of 0.06 unit in the Rfvalues of two conipounds was generally needed to obtain a clear separation in t n o spots. 2,1Dinitrophenylhydrazine itself shon s, in addition to the three spots (0.05, 0.32, and 0.71, Table I), one spot on the startVOL. 30, NO. 10, OCTOBER 1958
#
1621
ing line, the upper part of n hich gives a distinct green color b y spraying with aqueous 10% sodium hydroxide. The lower limit for obtaining this green spot was 5 y .
Physico-Chimique) for supplying a variety of derivatives used as test substances. LITERATURE CITED
R. J., Durrum, E. L., Zweig, G., Manual of Paper Chromatography and Paper Electrophoresis,” p. 237, Academic Press, New York, 1955. (2) Buyske, D. A., Owen, L. H., Wilder, P., Jr., Hobbs, M. E., ANAL.CHEW28,
(1) Bl:ck, ACKNOWLEDGMENT
The authors are indebted to M. Stoll, Director of Research, for his interest, to A. Saccardi for excellent technical assistance, and to colleagues in this and affiliated laboratories in Zurich (E.T. H.) and Paris (Institut tie Riologie
910 (1956). (3) Gaspari5, J., Verefa, M., Collection Czechoslav. Chem. Cmmun. 22, 1426 (1957).
(4) Horner, L., Kirmse, W., Ann. Chem. Liebigs 597,50 (1955).
(5) Lederer, E., Lederer, M:, “Chromatography, Review of Principles and Applications,” p. 170, Elsevier, New York, 1957. (6) Lynn, W. S., Jr., Steele, L. A., Staple, E., ANAL.CHEM.28, 132 (1956). (7) Meigh, D. F., Chem. & Znd. (London) 1956, 986. (8) S,chmitt, W: J., Moriconi, E. J., 0 Connor, I?. F., ANAL. CHEhl. 28, 249 (1956).
RECEIVEDfor review September 23, 1957. Accepted June 2, 1958.
Ion Exchange Chromatography for Hydrolysis Products of Organophosphate Insecticides F. W.
PLAPP and J. E. CASIDA
Department of Entomology, University o f Wisconsin, Madison, Wis.
b Many mono- and diesters of phosphoric and phosphorothioic acids can be separated by anion exchange and paper chromatography. Using Dowex 1 -X8, phosphoric, phosphorothioic, and mono- and dialkyl phosphoric acids are eluted with hydrochloric acid gradients. Methanol and acetone are used as cosolvents with acid gradients to elute dialkyl phosphorothioic, dialkyl phosphorod ithioic, monoa I kyl phenylphosphoric, and monoalkyl phenylphosphorothioic acids. These chromatographic techniques should be useful in investigations on the in vivo and in vitro degradation of organophosphate insecticides and related compounds.
0
insecticides are subject to in vivo and in vitro hydrolysis a t their ester and acid anhydride groupings. Both the site and rate of hydrolysis are major factors in determining the efficiency of the insecticide. The degree of hydrolysis of organophosphate triesters has generally been determined by partitioning with an organic solvent that will extract the unhydrolyzed phosphate, and leave the ionized hydrolysis products in the aqueous phase. The mater-soluble derivatives are then fractionated by forming insoluble salts (6) or by paper chromatography (7, 11, 12). K h e n present in biological fluids, it is often difficult to free these hydrolysis products from interfering compounds for chnmcterization and quantitative analysis. An anion exchange method for the quantitative determination of mixtures of hydrolysis products from organophosphate insecticides is drscribed. A paper chromatographic procedure for RGANOPHOSPHATE
1622
ANALYTICAL CHEMISTRY
4
0.8
YP
0.6
0.2
LITERS
Figure 1 . Ion exchange separation on Dowex I of metabolites of dimethyl 0-( 2,4,5-trichlorophenyl) phosphorothioate
0,O-
Reagents. I. Elution gradient p H 2 to p H 1 HCI II. Elution gradient p H 1 HCI plus methanol ( 1 :3) to 1N HCl plus methanol ( 1 : 3 ) 111. Elution gradient 1N HCI plus methanol ( 1 : 3 ) to concd. HCI, water, and methanol ( 1 : 1
ascertaining the purity of the fractions eluted from the columns is also reported. APPARATUS AND REAGENTS
The phosphorus compounds used and their sources are listed in Table 1. The resin employed was Dowex 1-X8 anion exchange resin, 100 to 200 mesh (enpacity 3.4 meq. per dry gram). Prior to use, the resin was R-ashed with 1 S hydrochloric acid to ensure its being in the chloride form, followed by distilled water, until the eluate was above pH 3. About 50 grams of the ccnditioned resin were slurried in water and poured into a column 2.5 cm. in diameter to a depth of 29 em. Gradient elution chromatography was used with pH 2 . pH 1, I-Y, and concentrated hydrochloric acid solutions as eluents. K h e n required, technical grade acetone and 99% methanol xere used as cosolvents. The gradient’ elution wzs achiwed by
:6)
placing the vieaker acid solution in a separator? funnel directly above the colLinin. A stronger acid solution was located in a second funnel of identical size ivhich 11-as positioned a t the same level as the first. The two funnels were connected by a siphon. As the weaker solution entered the column, the stronger acid was siphoned into the first funnel, giving a continuous inCreaSe in eluent acid concentration. A Ltream of air was ‘passed over the siphon inlet to ensure mixing of the t n o solutions. PROCEDURE
The column was prepared as described and placed on a fraction collector. The sample to be chroniatographed was dissolved in a small volume of distilled \later and pipetted onto the column. Sevcrd additional volumes of water n ere iwed to TT ash the sample into the
