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 20,N. Y.
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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
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 A N D 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-
pholipide isolated by alkaluie treatment ( 5 ) did not show significant movement in these solvents. Figures 1 and 2 give results obtained with lipide extracts of liver, intestine, and kidney of the rat. Similar effects were obtained n ith lipide extracts of the other tissues tested or with appropriate mixtures of purified phospholipides. Resultq obtained by circular chromatography of liver phospholipides in octanol-lutidine-acetic acid (45 to 2.5 to 5 ) are s1ion.n in Figure 1. The diagram illustrates a sector of the radioautogram and the spot tests used to identify the hands. Only the width but not the relative intensities of the bands is indicated. Bands 1, 2, and 3 were inteiise; band; 4 and 5 were weak. The chromatogram n a s run by Method A n i t h 60 y of rat liver lipide in 20 p l . of isoamyl alcohol-benzene (1 to 1). Radioautogram tracings were obtained in 2 6-dimethyl-4-heptanoneacetic acid (30 to 5) from lipide extracts of liver, kidney. and intestine of rats injectrd with radioactive orthophosphate (Figure 2). Caing Method B, 15 y of lipide evtract in 4 pl. of isoamyl alcoholbenzcw (1 to 1) nere applied. Bands 4 and 5 were intense. but 1 , 2 . and 3 lvere n r a k . Rand 3. C, was more intense than 2, C, or 1. C. The data in Figure 2 show that intestinal lipide had more lysolecithin than did kidney or liver. The solvent mixtures given in Table I provide systems for the separation of the various classes of phospholipitles froni each other and from the nonphospholipiclee. 2,6-Dinieth~l-i-h(>ptanone-acetic acid provided the bvst separation of the largest number of coniponents in a mi\turcl and the most clrw-cut separation of lecithin and cephalin. Lysolecithin was separated from lecithin in all the solvents. The br,st sepnratioii of lysolecithin and sphingoniyrlin n as obtained with chloroform-lutidine-aretic acid or octanollutidiiie-acetic acid. For best results, the concentration of individual phospholipicle or mixture must not be larger than the T.alues given in Table I and Figurrs 1 and 2 . The minimal amount of an individual phospholipide that can be detected by Method B using the dye test is about 1 y ; milch lms can be detected by radioautography if the lipides are labeled n it11 phosphorus-32. Ho~vever, a t these low concentrations, the lipides do not give the choline or ninhydrin tests previously described (5, 6). The solrent> previously descrihrd for
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Table
Compound Yeast lecithin Distearyl lecithin Egg cephalin Distearyl phosphatidj-1 et hanolamine Lysolecithin Sphingomyelin
I.
Rf Values of
Octanollutidineacetic acid, 45: 2 . 5 :5 0.50
0.49 0.35
Solvent System 2,g-Dimethvl- 3-Methyl-24-heDtanonebutanoneace'tic acid, acetic acid, 30:5 30:3 0.60 0.78 0.58 0.76 0.47 0.60
0 25
0.16 0.35
Figure 1 . Circular chromatography of liver phospholipides in octanollutidine-acetic acid (45 to 2.5 to 5) A. Rhodamine G dye test 6. Choline test C.
D. 1.
2. 3. 4. 5.
Purified Lipides
0.46 0 25 0.38
0.56 0.31 0 44
LITERATURE CITED
(1) Giri, K. V., Krishnamurthy, K.,
0.5i
0.54 0.53 0.15 0.35
Figure 2. Tracings of radioautograms obtained in 2,6-dimethyC4-heptanoneacetic acid (30 to 5) from lipide extracts of rats injected with radioactive orthophosphate
Ninhydrin test Radioautogram Nonphospholipide Lecithin Phosphatidyl ethanolamine Unknown Unknown
the separation of phospholipides with conventional chromatography on paper impregnated with silicic acid (3) also give excellent separations with circular chromatography. More clear-cut separations were obtained a ith circular than with conventional paper chromatography (5, 6). A smaller amount of phospholipide was required and the running time of the solvent mixtures was less. The technique of circular chromatography with the solvents described should be of particular value in qualitative experiments in which only minute quantities of lipide are available.
Chloroformlutidineacetic acid, 20:30:5 0.60
A. Liver 6. Kidney C. 1. 2.
3. 4. 5.
Intestine Unknown Unknown Lysolecithin Phosphatidyl ethanolamine Lecithin
Venkatasubramariian, T. A , , Lancet 263, 562 (1952). (2) Hack, H., Biochem. J . 54, 602 (1954). (3) Rlarinetti, G. V., Stotz, E., Riochim. et Biophys. Acta 21, 168 (1956). (4) hIarinetti, G. V., Witter, R. F., Stotz, E., J . Bid. Chem. 226, 475 11957). ( 5 ) Roiiser, G., Marinetti, G. V., Witter, R. F., Berry, J. F., Stotz, E., Ibid., 223.48.5 - - _ --- flR56) (6) Witter, R. F., Rlarinetti, I
\ - - - - / .
Morrison, A,, Heicklin, L., Biochem. Biophys. 6 8 , 15 (1957).
G. V., Arch.
RECEIVED for review November 15, 1957. Accepted June 2, 1958. Work supported by grants No. B-679 and H-2063 of the U. S. Public Health Service, National Institutes of Health, Department of H d t h , Education and Welfare.
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