plasmic component has not been adequately characterized cytologically. Whether it is identical with the “lysosome” fraction (3) is not certain; the distribution curve for acid phosphatase is similar to that of uricase (12) but not identical. The principal usefulness of this method has been the comparison of tissue of normal animals with those that have been treated in various ways. Thus enzyme distributions in livers of rats have been studied after partial hepatectomy (11) or carbon tetrachloride treatment ( I S ), livers of hypoxic guinea pigs (C), and livers of tumor-bearing mice (9). As an example of the applicability to other tissues, Figure 1 shows the distribution of cytochrome oxidase in the thymus of normal rats and rats sacrificed 1, 2, and 3 days after exposure to 600 r totalbody x-radiation. The size associated with maximum activity was decreased slightly but consistently in the irradiated tissue, and the shape of the distribution curve was markedly altered. The mechanism by which x-radiation produces this change is not altogether clear. DISCUSSION
Density-gradient centrifugation has provided a method for estimating the intracellular distribution of enzymes and other cellular components in terms
of the size of the cytoplasmic particulates with which they are associated. Such a procedure obviates the necessity of assigning morphologic labels to subcellular fractions of known heterogeneity. The method has certain drawbacks. Only a limited amount of material can be handled in one centrifuge tube; the author has used homogenategradient volume ratios of 1 to 10 or 1 to 12. It is obvious that the lower the ratio the better the resolution will be; and the smaller the amount of material available for analysis. The necessity for making certain assumptions about osmotic pressure gradients, sphericity of the particulates, particle density, and side-wall sedimentation has been mentioned. It is clear, also, that the homogenization process common to all differential centrifugation techniques, involving the disruption of cells by shearing and the liberation of cellular components into solutions of sucrose, is decidedly unphysiological. The author has used the procedure to measure the sizes of polystyrene latex spheres of 0.5 to 3.0 micron diameter. These experiments, carried out in collaboration with H. E. Kubitschek of this laboratory, will be reported elsewhere; very close agreement has been obtained between sizes of particles estimated by this procedure with those measured optically.
LITERATURE CITED
(1) hnderson, N. G., in Oster, G. and Pollister, A. W. (eds.), “Physical !l!echniques in Biological Research,” Vol. 111, pp. 300-53, Academic Press, New York, 1956. (2) Blaschko, H., Hagen, J. M., Hagen, P., J . Physiol. ( L o n d o n ) 139, 316 (1957). (3) DeDuve, C., Pressman, B. C., Gianetto, R., Wattiaux, R., Appelmans, F., Biochem. J . 60, 60.4 (1955). (4) Klein, P. D., Thomson, J. F., Am. J . Physiol. 187, 259 (1956). (5) Nichols, J. B., Bailey, E. D., in Weissberger, A. (ed.), “Physical Methods of Organic Chemistry,’] 2nd ed., Part I, pp. 676-9, Interscience, New York, 1959. (6) Schneider, W.C., J . Biol. Chem. 176, 259 (1948). (7) Tedeschi, H., Harris, D. L. , Arch. Biochem. Biophys. 58, 52 (1955). (8) Thomson, J. F., Klipfel, F. J., Zbid., 70, 487 (1957). (9) Thomson, J. F., Klipfel, F. J., Cancer Research 18, 229 (1958). 10) Thomson, J. F., Klipfel, F. J., Erptl. Cell Research 14, 612 (1958). (11) Thomson, J. F., Mikuta, E. T., Arch. Biochem. Biophys. 51, 487 (1954). (12) Thomson, J. F., Moss, E. M., Zbid., 61,456 (1956). (13) Thomson. J. F.. Moss, E. ?VI.,Cancer ’ 8; 789 (1955). ’ RECEIVED for review December 29, 1958. Accepted February 5, 1959. Based on material presented before the Division of Analytical Chemistry, Symposium on Separation Processes through Differential Migration Analysis, 134th Meeting, ACS, Chicago, Ill., September 1958. Work performed under the auspices of the U.S. Atomic Energy Commission.
Isolation of Carotenoids, Coumestrol, Chlorogenic Acid, and Antibiotics Application of Countercurrent Distribution C. R. THOMPSON, A. L. CURL, and E. M. BICKOFF Western Regional Research laboratory, Albany 7 0, Calif.
b
The application of countercurrent distribution technique to biologically active materials is reviewed briefly and examples showing its uses are given. These include carotenoids, isolated from oranges; a plant estrogen, coumestrol, isolated from Ladino clover; an isomer of chlorogenic acid from peaches; and the antibiotics, aterrimins, from Bacillus subtilis var. aterrimus.
T
isolation and identification of small amounts of biologically active constituents from plant or animal mateHE
838
ANALYTICAL CHEMISTRY
rials by the classic methods of extraction, precipitation, and crystallization are often next to impossible. Chromatography, or selective adsorption in the m-ider sense-column, paper, ion exchange-has served admirably with some substances, but the separation of similar entities by these techniques is often unsuccessful. Countercurrent distribution (CCD) may often accomplish the separation and, by prior knowledge of the different components’ behavior, also serve to identify each. This operation has been practical only recently, since the development of integrated and automatic
equipment for multiple transfers. Hundreds or even thousands of individual equilibrations are now possible within a few hours or days. This makes the high resolving power of the method usable for many separations and analyses. Craig and Craig (4)have been largely responsible for much of the development in this field. Compounds that have been resolved by countercurrent distribution include various glycerides in natural fats, steroid hormones, amino acids, protein fragments, organic acids, dyes, and chemical intermediates (10).
W
z
W
+
Figure 1. Countercurrent distribution of carotenoids from Valencia orange juice System. S k e l l y solve B-absolute methanol
0
a
7
4 v)
Q v)
P
4 0 c 4
Color was measured at 440 tnp and results calculated from conversion table forB-carotene
J
2
e
#
TUBE NUMBER
ADVANTAGES AND LIMITATIONS OF METHOD
In both countercurrent distribution and partition chromatography, a moving phase containing the material to be fractionated is passed along a stationary phase at such a rate that equilibrium conditions are approached. I n countercurrent distribution a stepnise transfer of the moving phase occurs; thus it can be thought of as “discontinuous partition chromatography.” The loner phase is stationary, 17 hile the upper phase moves in discrete steps as rapidly as the t n o phases will separate after each contact. The equilibrium distribution of each solute b e t m e n the two phases determines its retardation in passing through the train of tubes and consequently its final position a t the end of a distribution run. Countercurrent distribution is especially well adapted to the handling of unstable compounds. Changes in acidity or alkalinity are unnecessary. Any reagent added can be removed easily, usually under mild conditions. Practically everything can be recovered. If a particular solvent system fails to give the required separation, i t can be evaporated and another tried. Although prior knowledge of the nature of the compound sought is unnecessary to effectthe separation, knowledge of some property (chemical, physical, or biological) is required to locate the active fraction after separation. Some obvious difficulties are encountered. Poorly soluble, highly polar, or extremely nonpolar compounds may cause trouble. Proteins having molecular weights in excess of 6000 have not been successfully distributed. Selection of the proper solvent system is often the most difficult problem. A solvent system that breaks into two phases quickly is very desirable. Emulsions can be very troublesome; they may slow the operation or render i t impossible. Temperature changes may alter distribution coefficients markedly; for best results a constant temperature room is recommended.
Solubility data may be misleading for use in predicting distribution of a solute between two phases. Distribution in a partially saturated solution may be different from that in a nearly saturated solution. If a particular solvent system does not separate a pair of desired components completely, the initial distribution is carried out and all material is discarded except the tn-0 wanted fractions. The tubes are refilled and the last tube is connected to the zero tube. The system can now be recycled almost ad infinitum until the separation is achieved. This method is economical of solvents. requiring only the initial charging. Solvents must be free of residue or freshly redistilled. An excellent summary of mathematical models used in countercurrent distribution studies is presented by n’eisiger (10). TTorkers in this laboratory have made extensive use of countercurrent distribution in separating carotenoids, (6-7)) a plant estrogen (1, 2 ) , chlorogenic acid (S), and antibiotics (8). CAROTENOIDS
Carotenoids have been separated and studied for years by column chromatography Discrete fractions have been recovered by selective elution or extrusion of the column and separate elution of carved-out portions. Much was accomplished by these techniques, but separation of caroteqoids having h - 0 , three, and more hydroxy1 or epoxide groups per molecule 11-as less successful. The carotenes (hydrocarbons) are readily separated by chromatography after the xanthophylls are removed by a separation n ith petroleum ether and 90% methanol. The chromatographic separation of the xanthophylls is much less satisfactory. It 17-as found that countercurrent distribution could be used to separate the \anthophJ 11s into a number of fractions, nhich nere then much more readily separable b y chromatography. Therefore, countercurrent distribution has been exploited in studies of these pigments of citrus
fruits and other plant products ( 5 ) . The method has a n additional advantage, in that the appearance of a particular pigment in a given fraction suggests the nature of the functional groups, A simple system of methanol-Skellysolve B gave some fractionation of unsaponified carotenoids of orange juice (Figure 1); the saponified carotenoids were separated clearly into three portions. Before saponification, carotenes and fully esterified xanthophylls were found in fraction 1; after saponification, only carotenes remained. Fraction 11, after saponification, contained monohydroxy compounds such as cryptoxanthin. Fraction I11 contained diand polyhydroxy carotenoids and increased markedly as a result of saponification. I n these trials, samples containing up to 100 mg. of total carotenoids were used for distribution in the countercurrent distribution apparatus; 10 ml. of each solvent phase per tube was used. A larger amount of sample could have been used with the methanol-Skellysolve B solvent pair, because the phases separated easily, but other solvent systems emulsified badly when too much crude material was distributed. With benzene-Skellysolve B 4 7 % methanol a much more complete resolution was obtained ( 5 ) (Figure 2). The carotenes, cryptoxanthin, and a hydroxy-a-carotene-like substance were carried nearly to the last tube (fractions I and 11). Fraction I I I A was shown b y further chromatographic and spectrophotometric studies to be lutein and zeaxanthin, while I I I B m-as shon-n to contain five separate compounds-two isomers of antheraxanthin and three isomers of mutatosanthin. Fraction I I I C contained two isomers of violaxanthin, and three of luteoxanthin, plus auroxanthin. The slowest-moving fraction, IIID, contained valenciaxanthin, sinesiaxanthin, tm-o isomers of trollixanthin, valenciachrome, and trollichrome. Thus the primary separation of a very complex mixture 11-asachieved by countercurrent distribution. The technique was applied to elm leaves n i t h similar results. With leaf extracts the unsaponified extract caused too much emulsion for countercurrent distribution, except with Skellysolve B-99Y0 methanol. However, in this system chlorophylls obscured and contaminated the carotenoids. The presence of colorless hydrocarbons and alcohols also caused emulsions, but after saponification and partitioning between petroleum ether and 90% methanol the hypophasic fraction was distributed successfully (Figure 3). The diols were similar to those of orange juice, as was the small amount of monoether diols and the diether diols. The polyols were neoxanthin and isomers. VOL. 31, NO. 5 , MAY 1959
839
I
I
Diols
3GO-
Tube Number Figure 2. Countercurrent distribution of carotenoids from Valencia orange juice System. Skellysolve B-benzene-87% methanol
Table I.
Solvent System
-4 B C D
E F
Solvent Systems Used in Isolating an Estrogen
Solvents and Proportions by Volume B (10:5:5:2 Chloroform-carbon tetrachloride-methanol-73-ater (2 : 2 :3 :2) Methanol-benzene-ether-water
Acetone-ether-water-Skellysolve
No. of Transfers 100
Position of Estrogen in Tubes 69-90
100 100
56-80
100
25-58
30-60
(4:4:1:1)
Skellysolve B-ethyl acetate-methanol-carbon tetrachloride-water (1: 1: 1: 1: 1) Acetone-carbon tetrachloride-water (2: 1 : 1) Acetone-carbon tetrachloride-water-methanol (10:5:5:1)
COUMESTROL
Reports of abnormal response in animals pastured on Ladino clover have described nonnormal lambing in ewes, increased rate of gain in fattening lambs, and stimulated milk production in dairy cows. These symptoms suggested presence of a n estrogen in the plants. Assays employing immature female mice indicated high estrogenic activity in the clover. Fractionation of the plant material yielded a lipide extract which contained the estrogen, a very forbidding greenish black tarry mass. A preliminary purification of this crude concentrate was accomplished by extraction with dilute aqueous alkali, acidification of the aqueous extract, and re-extraction of the acid solution with ether. This treatment removed the great bulk of fat-soluble impurities and resulted in about 100-fold concentration of the estrogenic activity. However, the estrogen still represented only a few per cent of the dried weight of this purified concentrate. Application of countercurrent distribution n-as of utmost importance in finally obtaining a pure crystalline product. Successive application of five separate solvent systems was necessary before the pure material was obtained 840
Figure 3. Countercurrent distribution of hypophasic carotenoids from elm leaves System. Benzene-Skellysolve B-87% methanol
ANALYTICAL CHEMISTRY
100
40-76
280
30-60
(Table I). As much as 100 grams of concentrate was distributed in the large 100-tube countercurrent distribution apparatus which holds 100 ml. of each solvent phase per tube. To locate the position of the estrogen after a distribution, the contents of selected tubes taken a t intervals were evaporated to dryness, and a weighed aliquot mas fed t o immature mice. The increase in uterine weight plotted against the corresponding tube number of the fraction tested located the estrogen. A typical assay curve with solvent B is shown in Figure 4. Discovery that the estrogenic compound exhibited a strong bluish fluorescence under ultraviolet light aided the work immeasurably. Small samples from the countercurrent distribution apparatus were observed after migration on chromatostrips (9) or paper chromatograms. Following isolation of the estrogen i t ryas characterized and synthesized. The name coumestrol was assigned to the compound because of its coumarinlike structure, which differs from that of any of the previously isolated plant estrogens. It is about 30 times as estrogenic as the previously isolated plant estrogen, genistein.
I
20
,
40
1
60
1
80
1
,
I30
Tube Number
Figure 4. Countercurrent distribution of coumestrol from Ladino clover
CHLOROGENIC ACIDS
I n studies of the enzymatic browning of fruits a new isomer of chlorogenic acid, “neochlorogenic acid,” was isolated from peaches (S) by countercurrent distribution. A butanol extract of lyophilized peach puree was distributed between ethyl acetate and 23’ phosphate buffer, pH 3.0. Four fractions were obtained. The first consisted of anthocyanins plus polymeric materials; the second, neochlorogenic acid; the third, chlorogenic acid; and the fourth, polymeric unknowns. Removal of sodium ions on a n ion exchanger caused much loss of the neochlorogenic acid, but enough remained to obtain physical constants. This compound is thought to be coupled through the 1- or 4-position on the carbovylated ring. ANTIBIOTICS
Countercurrent distribution has also been employed to isolate two antibiotics from bacterial cultures. A pure material, Subtilin A, has been obtained from the peptide complex recognized as Subtilin some years ago. Snother antibiotic was obtained from Bacillus subtilis var. aterrimus and named “aterrimin” (8). Two compounds were recognized
in this complex as nonnitrogenous unsaturated lactones, Crude preparations of aterrimin are being sold for use in poultry feeds. These examples illustrate the value of countercurrent distribution in separating different. active materials from plant : r i d animal sources. As information accumulates, its use ,’ill, no doubt, tle exl,allded to serve many other purposes as well. ACKNOWLEDGMENT
The authors are indebted to J.
K.
Corse and J. C. Lewis for aid in assembling data on chlorogenic acid and antibiotics. LITERATURE CITED
E. RI., Booth, A. N., Lyman, 11. L., Livingston, A. L., Thompson, C. R., DeEds, F., Science 126, 969
( 1 1 Bickofl,
(195i).
( 2 ) Biclioff, E.
M.,Booth, A . S . , Lyman, R . I,., Livingston, A. L., Thompson, C. R., Kohler, G. O., J . A y r . Food Cheni. 6 , 536 (1958). ( 3 ) Corse, J. W.,S a t u r e 172, i 7 1 (1953). (4) Craig, L. C., Craig, D., “Technique of Organic Chemistry” (-4.Keissberger, ed.), 2nd ed., Vol. 3, Part 1, pp. 149-
332, Interscience, Sew Tork, 1056. (5) L . J~ . *‘g’. Food Che’71.‘ 7 466 ( 1953). (6) Curl, A . I,., Bailey, G. F., Food Research 22, 323 (1957). (7) Curl, A. L., Bailey, G. F., J . -4gr. Food Chem. 2 , 685 (1954). (8) Lewis, J. C., I b i d . , 1, 1159 (1953). (9) Lyman, R. L., Livingston, -4. L., Bickoff, E. &I., Booth, A. S . , J . Org. Chem. 23, 756 (1958). (IO) Weisiyer, J. R., “Organic .4nalysis,” Gol. 2, pp. 277-326, Interscience, Sew York, 1954.
RECEIVED for review Sovember 6, 1958. Accepted March 6, 1959. Mention of specific products does not imply endorsement by the Department of ilgriculture.
Examination of Oxyacids of Phosphorus by Electrochromatogra phy TAKUYA
R.
SAT0
Division of 6iological and Medical Research, Argonne National laboratory, lernonf, 111.
b Differential electrical migration in moist paper is an effective method for resolution of mixtures of the oxyacids of phosphorus. Certain separations, such as the isolation of pyrophosphate, have been improved by addition of chemical precipitants, as zinc salts, to the background electrolytic solution. The addition of radioactive tracers before migration and the use of neutron activation after migration have facilitated the location and estimation of the various oxyacids of phosphorus separated in the moist paper. Migration sequences, mixed migrations, and comparative migrations in the same sheet of paper have provided a convenient basis for identification of the acids separated from mixtures. Some common oxyacids of phosphorus separated by a single migration in acetic acid are, in order of decreasing mobility, trimetaphosphate, tetrametaphosphate, tripolyphosphate, pyrophosphate, hypophosphorous, phosphorous, phosphoric, and condensed phosphoric acids. This sequence also varied with variation of the solvents. The technique has been applied in tests for the purity of radioactive phosphorus tracers, and for detection of the alterations of phosphorus compounds exposed to heat or neutrons.
B
the importance of phosphorus compounds in nature (3, 36, 60, 65), analysis of mixtures of the oxyacids of phosphorus is essential to many nuclear (SR), chemical (19), and biological investigations (21, 26, 68). ECAUSE OF
Many chemical procedures that have been devised for the detection (7, 11, 28, 45,69), estimation (8, 9 ) , and separation (27, 35, 58,46) of the phosphorus oxyacids, including colorimetry (20, 54, 37), titrimetry ( 2 , 22, 44, 63), and precipitation (35, 42-44, 66), are not widely applicable to complex mixtures, particularly to those containing interfering substances. Differential migration methods such as paper chromatography have served for the examination of various oxyacids ( 1 , 13, Sf), some condensed acids ( I O , 15-17, 29, 59, 67), and certain phosphate esters (25), especially when supplemented by sensitive colorimetric methods for the detection of the separated substances (24). X a n y of these applications of the paper chromatographic method have been summarized recently by Hettler (2.4). ilnion exchange chromatography has also been utilized for the separation of many phosphorus compounds in acid solutions (6, 12, 25, 35). The assay of phosphorus compounds from physical properties such as u-ray diffraction (41, 45),viscosity (14, 58, 61), optical properties (4, OR), melting point (40, YO), and ultracentrifugation (SO) does not permit resolution of the mixtures. K i t h the exception of chromatography (anion exchange or paper), these methods provide very little information about the unknown constituents of mixtures or about the minor constituents. Promising separations of various oxyacids of phosphorus have been obtained b y differential electrical migration in lactic acid solution stabilized
with soft filter paper (39). This method has served for the examination of the neutron-activated acids (55, 57), for separation of phosphate, pyrophosphate, phosphite, and hypophosphite ( 5 4 , condensed phosphates from radioactive (tracer) phosphate (P3204) (60), and esters (47, Sg), and for demonstrating that slowly migrating condensed phosphates are formed by the neutron irradiation of phosphate (51, 52), by y-ray irradiation of phosphate, and by heating phosphate (55). The electrical mobility of the oxyacids varied with the hydrogen ion concentration (18, 48), buffer (48), and electro-osmotic transport (18). As a n extension of earlier studies (49, 50, 55, 57), electrochromatography has now been modified in several ways both for examination of the simple oxyacids of phosphorus and for examination of the complex condensed phosphoric acids. The distance of the electrical migration has been made t o exceed that usually produced by flow of solvent in conventional paper chromatography. This more extensive migration has facilitated the separation of the slowly migrating components of the mixtures. The background electrolytic solution has been varied and reagents have been added to improve certain separations and to stabilize some of the condensed phosphoric acids. Some of the reagents have been added as zones in the migration system (57), so that they transmit certain acids of phosphorus but retain others. These modifications of the method have made possible the separation and detection of VOL. 31, NO. 5, MAY 1959
841
