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 ( 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 by 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
various condensed phosphoric acids for which there are, as yet, no specific physical or chemical tests. These methods have facilitated the separation of the radioactive acids which were present in "carrier-free'' condition as well as those present in larger quantities. Because of the wide applicability and the great sensitivity of these electrochromatographic methods for examination of the oxyacids of phosphorus, the principal analytical techniques are described herein. Special applications of the techniques are being studied.
other shret of polyethylene n n J p I a 1 ~ 1 in tlic tlirrmal column of tlir r t w t i r for 2d3y9at a nrutrnn fluxof nppro\imntely neutrons pvr rq. ,:in. pw sccoiid. The paper was t l w n r i m o v d from t1.e rewtr,r :rnd pwtnittcd IO "cool" fr,r 5onw 7 to 11 drys IO r(rlurr the background $11 rhe paper itsdf. I t was t h i n rad:oaittogr:iplwl, using Kodik To Silnvn s-my tilm. With tliis acticttioii 7, d i . niquz, the I m c r limit of dctert.tl,ility c,i phosphorus in tllr pxpw uiis iuuii4 t o hc 0.001 7 of pl.oslil.r,rus i i i MI. of solutim.
RESULTS AND DISCUSSION
Studies of the migretion of phosphorus compounds were complicated by the complexity of the oxyacids of phosphorus. With reepect to the state of oxidation, these acids fall into two principal groups, fully oxidized and partially reduced. Within each group the acids may be classified with respect to the degree and nature of condensation (Table I). Sequences and separability were established with authentic,
EXPERIMENTAL
I n the anthor's electrochromatographic procedure, the mixtures of the phosphorus compounds were resolved by differential electrical migration from a narrow zone in moist filter paper. The resolved substances, separated as a series of zones, were located by spraying with molybdic acid followed by exposure to ultraviolet light (5) or by exposure of the paper to neutrons (65, 69) followed by scanning with a Geiger-Muller counter or by radioautography (60). The quantity of each component was estimated by counting techniques, and the individual constituents were recovered by elution of the active regions of the paper with water. The substances separated electrochromatographically were identified by comparison with authentic compounds submitted to electrical migration in the same sheet of paper.
For the determination of the migration behavior of the various phosphorus compounds, an apparatus was devised and the commercial filter paper purified by washing with acid (54). The filter paper sheets for the migrations were about 6 feet long (2 meters). For the migration studies, the paper was first moistened with 0.1M acetic acid. Aqueous solutions of the compounds (about 50 pl. of about 0.01M solutions) were placed in small zones on spots near the cathode end of the moist paper. The paper was then covered with the polyethylene sheets, and direct current potential, either 5 volts per em. for 17 to 20 hours or 10 to 15 volts per cm. for 2 to 4 hours, was applied. The ends of the paper were then cut off, and the paper was uncovered and allowed to dry in air. For the detection and location of the radioactive products, radioautographs were made with Kodak No Screen x-ray film. The separated substances were identified by reference to the migration or sequence of separate spots of authentic nonradioactive preparations, which were located by neutron activation, or alternatively, by the addition of radioactive tracers before the migration. For activation, the dried paper was placed on a sheet of polyethylene, and the two sheets were rolled together, providing a cylinder about 2 inches in diameter. This rolled paper was wrapped in au842
.
ANALMICAL
CHEMISTRY
Figure I . Radioautograph illustrating comparative electrochromatographic migration of oxyacids f r o m preparalions of inactive N a n H P 0 4 , I.. ^^ Na2HP08, NaH-PO?, NarP,O,, Na5P3010 ^^ 'I'
.
I
The inodive rubdances in the elestrochromatogrann were activated by exposure of the popsr to neutrons, ond the radioautograph was mod'e of this activated electtrochromoiogrom. Bockground sobtion 0.iM acetic acid, time 20 h I.
Oxyacids of Phosphorus Classified with Respect to Oxidation State and Degree of Polymerization
A. C#impletelv impletely oxidized forms 1. nfonomolecular Monomolecular phosphates Orthophosphate, €I,P04 Metaphosphate, HPOs 2. Condensed h o a r phosphates (P-0-1' bonds) Pyrophosplmte, H4P10i Tripolyphosphate, H,PaOlo Tetrapolyphosphate, HoP,Ou 3. Condensed cyclic phosphates (P-O-P bonds) Trimetaphosphate, (HPOI)3 Tetrametaphosphate, (HPO,),
B. Reduced forms 1. Monomolecular oxyacids Hypophosphite, H,P02 Phosphite, H,POJ 2. Condensed oxyacids ' 0.1' Imnds) ~yroplmptiite,H,P,Oa Isohypophosphate, H4PlOr 3. Condensed oxyacids (P-P bonds) Hypophosphatr, H,P20s Diphosphite, H,P20r
Figure 7. Radioautograph illustrating electrochromatographic examination of H3P3z04purified by ion exchange and heated in a beaker on a hotplate a t 270' far 0.5 hour Dark o r e w represent locations of rodiomctive products. Hatched circles represent positions of (I"thentic acids of phosphorus added as 0.01 M solutions of their m i l s and located with rnolybdote reagent after the migrotion
in the preparation and handling of the oxyacids of phosphorus. When solutions of radioactive phosphate, purified by anion exchange chromatography, mere evaporated, even a very short overheating of the residue producrd a variety of condrnsation products. These products, separated and identified by differential electrical migration, proved to be pyrophosphate and other condensation polymers (Figure 7). These investigations show that elmtrochromatography is a remarknhly sensitive and effective technique for the examination of oxyacids of phosphorus. It serves for the resolution of mixtures, purification of tracers, identification of the various substances, and detection of the reactions produced by various conditions, such a8 heat, oxidation, and neutron irradiation. ACKNOWLEDGMENT
The author cxprcsses appreciation to H. H. Strain for advice and encouragement in the dcvclopment of thrse methods. These studies were also aided in a material way by John R. Van Wazer, Peter G. Arvan, and Edward J . Griffith, Monsanto Chemical Go.; by Russell N. Rrll and Lowell E. Nethrrton, Victor Chemical Works; by Oscar T. Quimhy, Procter and Gamble C o . ; Therald Moeller, University of IIlinois; by Ernest Leininger, Michigan State University; by B. Raistriek, 844
ANALYTICAL CHEMISTRY
Scottish Agricultural Industries, LM., Edinburgh, Scotland; and by Bruno Blaser, Hcnkel Co., Diisseldorf, Germany. all of whom contributed special phosphorus compounds in this rrsearch. The author is indebted to members of the staff at the Argonne Reactor for supervising the neutron irradiations and to Thomas Doody for assistance in constructing a vacuum dehydration apparatus. LITERATURE CITED
(1945).
( 5 ) Bandurski, R. S., Axelrod, B., J . B i d .
Chem. 193, 405 (1951). (6) Barney, D. L., Gryder, J . M',, J . Am. Cl~.am.Soe. 77, 3195 (1955). ( 7 ) Bnrtan, C. J., ANAL.CHEM.20, 1068 I1.Q4Ri .. .-,. ( R i Brll, R., Im. ENG.CHEM.,ana^. ED.
,
"'Y',.
>allis,C. E., Van Waaer, J. R., Met-
mlf, .I. S.,J . Am. Che,,i. Soc. 77, 1468 (1955). (15) Croxther, J., ANAL.Cmbi. 26, 1383 (1954). (16) Ebol, J. P., Ai'ikmchbri. i l c l a 6, Gig (1957). (17) IChel, J. P., Volmar, Y.,Cowpt. Tend. 233, 415 (1951). (18) Engelke, J. I,., St,rain, H. H., . ~ N A L . CHEM.26, 1872 (1954). (19) Fiskrll, J. G. h., Dchig, 11'. .4., Oliver. W. F.. Can. J . Chrtn. 30. 9 (1052 j. (20) Gnssner, K., Afikrochiw. -4cla 3 4 , 594 (1!)57], (21) Gossclin, R. E., Xegirian, R., J . Phanarol. Eznll. Therm. 115. 402 (1955). (22) Griffit,h,E. J., ANAL.CHEM.28, 528 (1956). (23) Harrap, F. E. G., Sntilrc 182, 876 (1958). (24) Hettlor, H. J., Ch7osmtogrnphy 1,389 (1958). (25) Higgins, C. E.i Bnldwin, 11'. H., ANAL.CHEM.27, 1180 (1955). (261 Ingelman, B., Malmgren, K., Acta Chem. Smnd. 4, 478 (1950). (27) Joncs, I,. T., IND.Es-G. CHEM.,ANAL. Eo. 14, 536 (1942). (38) Jones, R. T., Rvift., E. H., ANAL. CHEM.25, 1273 (l!l53). (21)) K;wl-Kronpa, E., Ibid., 28, 1001
(53) &to, T. R., Sorris, \I-. F.> Strain, H. H., Abstracts of Papeis, p. 43, 124th meeting, .ICs, Chicago, Ill., 1953. (54) Sato, T. R., Norris, IV. P., Strain, H. H., Ahr.4~.CHEL 27, 521 (1955). (55) Sellers, P.A , , Sato, T. It., Strain, H. H., J . IrkOTg. and 1 7 u c k i ?Chem. 5, 31 (1857). (56, Strain, H. H., Sato, T. R., A 4 x . \ ~ . CHEN.28. 687 11956). (57) Strain,”. H:, Sato, T. R., conference on use of isotopes in agiiciilture, p. 175, IT. 8. -4tomic Energy Comm., TID-7512 (1956). (58) Strauss, T’. P., Trwtler, T . I-..J . Am. C’hern. SOC.77, 1473 (1065).
(59) Thilo, E., Grunze, J., 2. anorg. u . allg e m . Chenr. 290, 209 (1957). (60) Van \Yazer, J. R., “Encyclopedia of Chem. Technology,” T’ol. 10, Kirk and
Othmer, ed., p. 413, Interscience, 1053.
161) 1-an Wazer. J . R.. J . A m . Chenr. SOC. ‘ 72, 906 (1950)’. (62) Tan \Taxer, J. R., Goldstein, AI., Farber, E., Ihid., 75, 1503 (1953). (63) Tan TYazer, J. R., Griffith, E.J., RIcCulloueh. J. F.. A x . 4 ~ .CHEX 26. 1755
11854’l.” ,- - - , ‘ (64) Wade, H. E., Aforgan, D. >I., Biochem. J . 60, 264 (1955). (65) Waggaman, \I7. H., “Phosphoric Acid, Phosphate and Phosphatic Ferti-
lizers,” 2nd ed., Reinhold, XeTv York, 1952. (66) FTeiser, H. J., Jr., . ~ . ? - A L .CHEJI. 28, 477 (1956). (67) Restman, -4. E.R., Scott, A. E., Pedley, J. T., Cheniist,y in Can. 4,35 (1952). (68) +Kiame,J. >I , J . Bzol. Chem. 17, 919 (1949)
\ - - - - / .
(69) Wnteringham, F. P. IT., Harrison, A , , Bridges, R. G., Analysf 7 7 , l Q(1952). ( 7 0 ) Zettlemover, -4.C., Schneider, C. H., ’ Anderson, H , I-,, Fuchs, R. J.. J . Am. Chem. SOC.61, 991 (1957). RECEIVED for reviexv January 12, 1959. Accepted February 26, 1959.
Separation of Antibodies by Starch Zone Electrophoresis PETER STELOS‘ and W. H. TALIAFERRO Department o f Microbiology, University of Chicago, Chicago 37, 111.
b Rabbit antisera against sheep red cells were fractionated by starch zone electrophoresis to ascertain the distribution of antibody in the serum proteins and ascertain if the number of antibody species was dependent on the immunization schedule. In antisera obtained early in immunization the antibody activity migrated as a single peak between p- and y-globulins and sedimented with a globulin fraction of molecular weight about 1,000,000; late in immunization a second antibody migrated in the more slowly moving y-globulins and sedimented with a fraction of molecular weight about 160,000. Physically distinct antibodies of the same serological specificity may be induced by a given antigen and the ratio in which they appear i s a function of the immunization schedule.
Z
ELECTROPHORESI~ in a starchsupporting mcdiuni is usefiil in resolving a complex mixture of proteins into its individual components. The chief advantages of the method over other preparative electrophoretic methods are: the relatively large amounts of material that can be separated in a single operation, and the comparative ease and simplicity of the experimental procedure involved. As with all electrophoretic methods, the degree of resolution attainable depends on the nature of the proteins involved and, particularly, the net electrical charge that these bear in a buffer of given p H and ionic
strength. Since its introduction by Kunkel and Slater ( 4 ) to study the lipoproteins of blood qeruni. the method has been used in the analysis of rirus inhibitors ( I O ) , bacterial enzymes ( 6 ) , antibodies against diphtheria toxoid @), serum proteins ( I ) , and many others. The work reported here involved the characterization of rabbit antibodies against sheep rcd blood cells formed after inimuniaation with sheep red cells
or other material such as guinea pig kidnry. These antibodies, hemolysins, have the property of lysing sheep red cells when mixed Ivith complement. a normal serum component. Hemolysins are particularly useful in immunological studies, because the amount of hemoglobin released by the lysed red cells can be measured aecuratelJ- and conveniently by photometric methods. I n general, hemolysinf of two types occur; one has a molecular weight of
-300
osc
Present address, Sterling Chemistry Laboratory, Yale University, New Haven, Conn.
p-s
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B ___---
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5
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-
Figure 1.
Zl
ro I ‘c r ~
-
21
v
23
rkrwi/si
3’+
31 IO
tiny
41
-I 41
+‘o,nr
Starch zone electrophoresis of a normal rabbit serum
In this and all other tlgures electrophoresis was performed in pH 8.6 veronal buffer, 0.1 ionic strength (7)
VOL. 3 1 . NO. 5, M A Y 1959
845
