ing line, the upper part of II 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 pH 2 to pH 1 HCI II. Elution gradient pH 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 acliiwed 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
resin and t o remove any nonionized materials from the column. Excess supernatant was drained from the column until the liquid was 1 cm. above the level of the resin bed. The elution gradient apparatus was attached and the chromatograph developed. -4pproximately 20-ml. fractions n-ere collected a t the rate of about 3.5 minutes per fraction. Then 1-ml. aliquots were analyzed for total phosphorus by the method of Allen (1). T h e r e metliaiiol or acetone was used, the sqiiple was made alkaline to phenolphthalein with sodium hydroxide, and the organic solvent evaporated before perchloric acid oxidation. Paper Chromatography. T h e purity of the known compounds and of fractions eluted from t h e columns m r e deterniiiied using ascending, single phase, paper chromatography. Strips of J'ihatman KO.1 paper 5/8 X 7 inrhes were used. The compounds w r e spotted on the paper 2 em. from the bottom and the chromatographs developed to a height of 14 to 15 em. in 1 X 8 inch test tubes. The solvents listed in Table I pvere based on previously reported syst,ems (4, 7 ) . The phosphorus derivatives were located on the developed chromat,ographs by the niet'hod of Ilanes and Isherwood (j)using , ultraviolet light as t8he reducing agent. A second chromogenic agent' was used to locate the phosphorothioate compounds. The papers were sprayed with a 2% solution of cupric chloride, and then \yith an 0.57, solution of potassium ferricyanide, giving red-brown spots on a yellow-green background. Both methods were equally sensitive and gave definite spots with as little as 1 y of compound. RESULTS
Table 1.
Compounds Studied and R, Values with Two Paper Chromatographic Systems R/ Values, Temp. 25-28' (2.
Compound ( HO)zP(0)OHa ( HO)zP(S)OHC ( CHIO )P(O)(OH) z d ( CzHs0 )Pi0 OH ) z d
(i-C3H70)P(0)(OH)zd (n-C4HsO)P(0)(OH)zd ( CH,O)zP(0)OHd (CJLO)zP(O)OHd (Iso-CaH70)zP(0)OHd (n-C4HeO)zP(0)OHd (CH,O)ZP(S)OH' (CHaO)ZP(S)OKe ( CzHsO )zP(S)OH' (CzHs0 )zP(S)OK' (CH,O)*P(S)SH' (CH,O)zP(S)SKe (CzH,O)zP(SPH' (C&O)zP(S)SK' (CHaO)(HO)P(O)O+CI3-2,4,5Q ( CH,0)(HO)P(S)0+C1r2,4,5h a As monopotassium salt. 6 Standard deviation. c -1s trisodium salt ( I O ) . d Victor Chemical Works.
2-Propanol, "*OH,
2-Propanol, HZO, "40H,
75:25 0.00 0.00 0 . 0 4 i0 . 0 1 0 07 i 0 . 0 1 0 08 i.0.01 0 1 6 i O 02 0 . 4 4 f0 . 0 1 0 . 6 1 zk 0 . 0 1 0.74 i0.02 0 85 f 0 02 0 65 1.0 02 0 59 zk 0 03 0.77 i0.02 0.73 i0 . 0 1 0.74 f 0.03 0 . 6 8 i.0.03 0 . 8 9 i 0.02 0.84 f0.02 0 . 7 9 zt 0 . 0 1 0 . 8 6 i 0.02
75:24:1 0 . 0 5 f 0.01* 0 . 0 3 f 0.00 0.10 =t 0.01 0 . 1 5 =t 0.01 0 21 1 0 . 0 2 0 . 3 1 =t 0 . 0 4 0.59 1 0.03 0.76 1 0.01 0 . 8 8 i 0.04 0 95 zk 0.01 0 73 f 0 03 0 67 i 0 01 0 . 8 7 =t 0.03 0.79 1 0.02 0.85 1 0 . 0 2 0.78 f0.03 0 . 9 4 =t 0.02 0 . 8 7 i0 . 0 4 0 . 8 2 1 0.02 0.96 f 0 . 0 2
e f
h
ilmerican Cyanamid Co. Hercules Powder Co. .Is potassium salt ( I O ) . As ethylamine salt, Dow Chemical Co,
.I
0.8
AND DISCUSSION
The order of elution of the phosphoric acid derivatives from the ion exchange column was a function of both the primary p K value and the effect of solvent action. K i t h the varying alkyl derivatives studied (Table I), the position of elution from the columns was not materially affected by the nature of the alkyl group. Elution of phosphoric, nionoalk!-l, and dialkyl phosphoric acids occurred in the order of their increasing acid strengths (Figure 1). Of the alkyl groups studied, the methyl derivatives are the strongest acids, followed by the ethyl, n-propyl, and n-butyl, the nionoalkyl acids being slightly weaker than the corresponding dialkyl derivatives (9). The ion exchange system used did not adequately separate phosphoric, mono-, and dialkyl phosphoric acids, but paper chromatography provided a method for positive identification u-here mixtures were present. Phosphoric acid could also be differentiated from the alkyl phosphoric acids by analysis of the eluted fractions for free orthophosphate and for total phosphorus follon ing oxidation I\ ith perchloric acid. The
Figure 2. Ion exchange separation on Dowex 0,O-dialkyl phosphorodithioate insecticide
I of metabolites of an
Reagents. I. Elution gradient pH 2 to pH 1 HCI II. Elution gradient pH 1 to 1N HCI Ill. Water to bring eluate above pH 1 IV. Elution gradient pH 1 HCI plus methanol ( 1 : 3) to 1 N HCI plus methanol (1 : 3) V. Elution gradient 1 N HCI plus acetone ( 1 : 3) to concd. HCI, water, and acetone ( 1 : 1 : 6) VI. Concd. HCI, water, and acetone (1 : 1 : 6 )
difference represented the mono- and dialkyl phosphoric acids. I n the case of the phosphorothioic and phenylphosphoric acid derivatives, stronger acid conditions and solvent action were necessary for satisfactory elution. Phosphorothioic acid was eluted v, ith hydrochloric acid alone. The use of methanol and acetone as cosolvents was necessary for satisfactory elution of the dialkyl phosphorothioic and phosphorodithioic acids (Figure 2). Cosolvents were also required for elution of the monoalkyl phenylphosphoric and phenylphosphorothioic acids studied (Figure 1). The use of alcohols to elute highlj sorbed phenolic compounds
from this type of resin has been reported ( 2 ) . When known amounts of the organophosphate derivatives studied were chromatographed individually on ion eschange columns, they were quantitntively eluted in a single peak. HOMever, many of these derivatives are unstable in strong acid solutions (8) and if recovery of the eluted fractions is desired without appreciable degradation, they should be quickly extracted from the column eluate with amyl alcohol, various higher ethers (S), or chloroform. The method has been applied in this laboratory to studies of the in vivo and in vitro hydrolysis of phosphate and VOL. 30, NO. 10, OCTOBER 1958
1623
phosphorothioate insecticide-. Phosphorus-32-labeled insecticides have been administered to mammals, plants, and insects, and the hydrolysis products formed identified by cochromatography with known derivatives, or b y infrared spectra obtained on the eluted derivatives. Several organophosphate in vivo hydrolysis products other than tho-e reported hare been isolated by this ion exchange method and identified. The method is also useful in the purification of various primary and secondary esters of phosphoric and phosphorothioic acids. ACKNOWLEDGMENT
The authors wish to express their ap-
preciation to R. N. Bock and E. H. Wiegeshaus for advice and assistance. LITERATURE CITED
(1) Allen, R. J. L., Biochein. J . 34, 858
(1940'1. ( 2 j Anderson, R. E., Hansen, R. D., Ind. Eng. Chem. 47, 71 (1955). (3) Crandall, H. W.,Stewart, D. C., U. S. Patent 2,658,909 (Nov. 10, 1953). (4) Eble, J. P., Mikrochim. Acta 679 (1954). (5) Hanes, C. S., Isherwood, F. X., Satitre 164, 1107 (1949). (6) Hartley, G. S., Heath, D. F., Zbzd., 167, 816 (1951). ( i ) Kaplanis, J. 1 ., personal communication. (8) Kosolapoff, G. RI., "Organophoa-
phorus Compounds," p. 232, Kiley, Yew York, 1950. (9) Kurnler, W. D., Eiler, J. J., J . Am. Chem. SOC.65, 2355 (1943). (10) Plapp, F. W., Casida, J. E., J . Agr. Food Chem. 6, 662 (1958). (11) Robbins, W. E., Hopkins, T. L., Eddy, G. W.,J . Agr. Food Chein. 5, 509 (1957). (12) Robbins, W. E., Hopkins, T. L., Eddv. G. IT., J . Econ. Entomol. 49, 801 -(1956). RECEIVEDfor review January 6, 1958. Accepted May 26, 1958. Supported in part by the Research Committee of Graduate School from funds supplied ,by Wisconsin Alumni Research Foundatlon and by a grant from The Doiv Chemical Co. Approved for publication by the Director of the M-isconsin Agricultural Experiment Station.
Circular Chromatography of Phospholipides ROBERT
F. WITTER,1 G. V.
MARINETTI, LlLlAN HEICKLIN, and MARY A. COTTONE
Department o f Biochemistry, University o f Rochester School of Medicine and Dentistry, Rochester
-
-
2,6 Dimethyl - 4 heptanoneacetic acid (30 to 5), 3-methyl-2butanone-acetic acid ( 3 0 to 3), octanol-lutidine-acetic acid (45 to 2.5 to 5), and chloroform-lutidine-acetic acid (20 to 30 to 5) have been found to b e good solvents for the circular chromatography of the phospholipides. Excellent separations have been obtained both with mixtures of purified phospholipides and with phospholipides isolated from various tissues of the rat.
S
OLVENT SYSTEMS for
the separation of phospholipides on acid-washed unimpregnated paper were reported recently from this laboratory (5, 6). These solvents a e r e found to give even better results when the technique of circular chromatography was employed, and this publication describes the experi. mental conditions and the results obtained using this technique. Experiments were conducted with mixtures of purified lipides and with lipide extracts of liver, kidney, spleen, intestine, and lung of rats which had been injected Kith radioactive orthophosphate. Only one other report (2) has appeared of the circular chromatography of phospholipides. EXPERIMENTAL
The method of preparation of the lipide extracts, the administration of the 1 Present address, U. S. Public Health Service, Communicable Disease Center, Technology Branch, Box i59, Savannah, Ga.
1624
ANALYTICAL CHEMISTRY
radioactive orthophosphate to the rats (4), the sources and purity of the reference lipides ( 5 ) , and the tests for the detection of the phospholipides (5, 6) have been described. K h e n chromato grams were run by the latter of the following methods, four samples coukl he run on the same paper.
A. Ten to 20 p1. of isoamyl alcoholbenzene (1 to 1) (v./v.) containillg 15 to 30 y of a n individual phospholipide. or 60 to 80 y of a tissue lipide extract were placed in the center of a circle of acid-washed Whatman S o . 1 filter paper which was 15 cm. in diameter. B. A circle, 3 cm. in diameter, v a s drawn a t the center of the paper, and 4 pl. of isoamyl alcohol-benzene containing from 3 to 5 y of a n individual phospholipide, or 15 to 20 y of a tissue lipide extract, were placed as a curved band along approximately 90' of the circumference of the 3-cni. circle. After a wick of paper was attached via a pinhole in the exact center of the paper ( I ) , the circular filter paper was placed on top of a glass pie plate .rvith the wick dipping down into the solvent which had been placed in the dish to a depth of about 1 cm. The pie plate was a borosilicate glass dish about 15 cm. in diameter and 2 cm. in depth. The bottom of the dish was flat, and the rim ITas made of flat ground glass. 1 cm. in width. Then another pie plate was inverted and set on top of the paper. The two pie plates were put on a glass plate, and an inverted crystdlizing dish was placed over the pie plates and sealed to the glass plate s-ith a grease such as Lubriseal. Between 1 and 2 hours were required
20,N. Y.
a t 21-3' C. to develop the chromatogram, with the exception of the octanollutidine-acetic acid solvent which required 5 hours. After the papers were dried in the hood a t room temperature for 4 to 6 hours, they were washed with distilled water and again dried in the hood until dry to the touch. Appropriate sections of the paper n ere used for the following spot tests: Rhodamine CT (general lipide test), ninhydrin test (lipides containing amino groups), and phosphomolybdate test (lipides containing choline) (5, 6 ) . Detection of the developed bands was facilitated by observing the paper with the light re flected from a white surface. Radioautography (4) was used also to detect the phospholipides in lipide extracts of tiswe of rats injected with orthophosphate. The composition of t h r solvents is given on a volume-to-volume basis. The sources and properties of the solwitq have heen described (5. 6).
RESULTS AND DISCUSSION
The R , values obtained n-ith representative purified lipides are given in Table I ; they are similar to those found by means of conventional paper chromatography ( 5 , 6 ) . The phospholipides ere run as described in the esperimental section (Method A or B). The non~~liospliolipicles were run a t a concentration of 100 y per 20 pl. Cholesterol. cholesterol palmitate, ceramide. tri-stearin. monopalmitin. oleic acid. stearic acid, and phosphatidic acid had R , values grwter than 0.9. Acetalphos-
