Analysis by Electromigration plus Chromatography H4ROLD H. STRAIN
AND
JAMES C . SULLIVAN
Argonne National Laboratory, Chicago, Ill. This investigation was undertaken to provide w idelj applicable analytical methods for continuous operation and of potential industrial importance. To this end, electromigration was combined with various chromatographic techniques. With flow of electrical current at right angles to the flow- of solvent in a special cell, mixtures of various ions wrere separated completely and continuously. With this method
one ion could be substituted coiitiuuously for ailother, and in the presence of complexing agents that affect the adsorbability and the ionic mobility of various solutes, many different kinds of substance5 could be isolated and detected. JIodifications provide sensitive and rapid discontinuous procedures for the separation of mixtures of ionir wbstancec, as in qualitative analysis.
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plates. Cells designed for rontinuouo operltcion were Constructed of paper with a paraffined central compartment as indicated by Figure 2. Cells designed for discontinuous operation Kere constructed without this compartment. compartment .D,D estrntiing continuously from side to side. In both types of cells, the adsorptive imacerial was commerc.ia1 filter paper (0.1 inch or 2.5 mm. thick). Two papers (Filpaco, No. 046, The Filter Paper Co., Chicago, 111.; and Grade 320, Eaton-Dikeman, Mt. Holly Springs, Pa.) gave reproducible results and exhibited esceptionally high filtration rates. Both papers ocrasionally contained iron and copper in small spots (prohably in the form of oxides). For preparation of the cells, the aides of the paper and tht. ('entral compartment were brushed nith a saturated solution of paraffin in carbon tetrachloride or in petroleum ether (boiling range 65' to 110' C.), and the saturated areas were allowed to drj-. With paper designed for the continuous procedure, the dried, central paraffined area was cut with a sharp razor blade, and a narrow strip of untreated filter paper (Eaton-Dikeman, Grade 625, thickness 0.030 inch) was inserted as indicated by the figure. The inserted paper served as a wick for the continuous addition of a narrow zone or stream of the nlixture to be resolved. For this purpose. the common analytical papers also proved usPt'u1.
IAECTROMIGRATIOK ( 2 , $, 10, 12) and chromatography (3, 15, 17, 19, 20, 2.4) have long been employed for the resolution of mixtures of solutes. In 1939, Strain utilized electromigration, without flow of solvent, for the separation of dyes in a column of moist porous adsorbent (21). Under the name of electrochlariiatography, this method has been widely applied to the resolution of mixtures in C O I U ~ I ~and L : in strips or stacks of filter paper (3, 8, c), 13-16, 93, 26). It hae proved effective with charged partirles in diqxrsed system; a s well as u ith the inorganic ions (6, 8, 23, 25)
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Figure 1. Separation of 0.02 i M Methyl Orange and 0.02 M Phenolphthalein Separated by upward flow of wash liquid, W L (4 M ammonium hydroxide), and by horizontal flow of electric current. Paper (Grade 301 or 625,0.030 inch thick, Eaton-Dikeman), clamped between plastic plates t o prevent evaporation of ammonia. 300 voltq, 2 5 t o 35 ma.. 1 hour
In 1948, Haugaard and Kroner employed electrical migration plus simultaneous flow of solvent for the discontinuous separation of ionized substances in a sheet of paper, the current flowing at right angles to the flow of solvent (11). -1modification of this method, which requires only about an hour for resolution of a mixture, is shown in Figure 1. In 1949, Svensson and Brattsteii utilized electrical migration plus simultaneous flow of solvent for the continuous separation of dyes in a cell filled with pov-dered glass on which there was no adsorption of the ionized solutes (22), and Strain designed an :inalogous analytical system for the continuous separation of solutes by electrical migration plus chromatographic adsorption (18). An elaboration of this latter method forms the basis for this report.
Figure 2.
Electlographic Cell for Continuous Operation
.i. Filter paper B . Regions of paper impregnated a i t h parafin Outline of glass plates D. Compartments for addition of 7Yae.h liquid E. Compartment for addition of solute mixture F . Paper wick through paraffined region G . Platinum electrodes in grooves in glass plate H . Paper strips t o promote iiniform flow of wash liquid C.
4PPARATUS
Most experiments summarized here %-ereperformed in electrographic cells constructed of adsorptive paper held between glass
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V O L U M E 23, NO. 6, J U N E 1 9 5 1
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This prepared paper wab placed between two glass plates ab indicated by Figure 2. Platinum electrodes were inserted in grooves ground in one of the plates, and the plates were pressed to the paper with screw clamps. This electrographic cell was clamped in an upright (vertical) position between two ring \tands. A pan for the collection of the effluent m-as placed under the cell, and separatory funnels for the continuous addition of \\.ash liquid were supported on the ring stands. COKTJAUOUS OPERATION OF THE CELLS
DISCONTlNLiOUS OPERATION OF CELLS
For continuous operation of the electrographic cells, wash liquid \hatremained fixed in the paper. One example is t,he precipitation of metallic mercury by treatment of mcrcurous mercury ivith aqucous ammonia (Figure 27).
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20, both electromigration and differential adsorption determine the paths in the electrographic cell (Figure 23). Under these conditions, the ions will always follow separate courses, even though electromigration and chromatopaphic migration should play equivalent roles for both ions. Determination of the relative elect8ronligrationand chroniat,ographic rates of silver and of copper inns under the conditions represented by Figure 10 yielded the results summarized in Figure 23. In this figure, the chromatographie migration of the copper ions in the paper strip was much less t,han the migration found in the electrographic cell, an effect attributable to a slowly revwsible reaction between the copper ions and the cellulose. In some of the cont'inuous separations, the concentration of t,he ions MBS increased in order to determine the effectiveness of the electrographic cells. With ferric and nickel ions in the presence of tartrate and ammonia, the separations were effective with solutions 0.2 111 with respect to each cation. With increasing roncentration, the courses folloived by the ions became wider and their sepnration decreased.
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Figures 14 to 19. Continuous Separations 14. Ferric a n d aluminum nitrates, each 0.005 AI in W L , ca. 0.005 M dimethylglyoxime and 0.01 -1.1 tartaric acid in 4 .I1 NHrOH. R , aluminon in ca. 50% acetic acid. 250 volts, ca. 90 ma. 15. 0.005 M dimethylglyoxime, 0.0001 M methyl orange, and 0.005 M dichromate. W L , 0.1 M acetic acid. R , nickel plus ammonia. 250 volts 30-32 ma. 16. Stannous, arsenious and antimonous chlorides, each 0.005 M (n W L ,0.02 M lactic acid, 0.02 M tartaric acid, and 0.04 41 dl-alanine. R HIS. 300 volts, 95 ma. 17. Silver, nickel, copper, and ferric nitrates, each 0.005 IM in W L 0.01 M disodium tartrate a n d 0.01 .V ammonium oxalate in 4 M "a0H R dithio-oxamide a n d HxS. 160 volts, over 100 ma. 18. Silver a n d nickel nitrates, eaoh 0.005 in W L 4 M NHaOH. R , dithio-oxamide a n d diphenylthiocarbazone. 160 volts, 60 ma. 19. Silver and nickel nitrates, each 0.005 M in W i , 0.01 M ammonium oxalate in 4 .W KHaOH. R , dithio-oxamide and diphenylthiocarbazone. 160 volts, over 100 ma.
V O L U M E 23, NO. 6, J U N E 1 9 5 1
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.hiother esaniplr is tilt, precipitation of nickel by dimethylglyoxime contained in the :immoniacal wash liquid (Figures 28 arid 30). Under these conditions, the principles of chemical prwipit:rtion, electromig:ition, and chromatographic separation were utilized simultaneously. Ions surh as silver aiid nicakel, which were inseparable under the conditione of continuoiLc flow (Figure 18), xvere separated quickly I)>- the diwontinuous pi,o(:edurc (Figure 24). Ions of copper and nickel, likewise insrp;irnhlr hy the continuous procedure when animoniiim acetate in coiicentrntcd nmmonium hyclrosidc was thc n.ash liquid, were qiiic.l\ly w1):11’atrd1)y the discontiniiow procdurtt (Figure 25). 111 t h c s presence of cuiiil)lt~s-fo~iniiig reagents, most ions niigrutcd Its a single zonv in the elc~~troniigration and chromat,ographic systems. An exception to t,his relationship ~x-asobserved \\.it11 cobaltous ions that wcw added to a solution of t,artaric: : i d i n :iqueow ammoniti :inti :illon-ed to stand vith :iir for several 1ii)ws. Kith these oxidized solutions, both anionic anti caationic. forins of cobalt were squratrd 11y electromigration. In :widic s ~ l u t i o n i;oh:tlt ~. nncl nickel ions were slowly separable by c.lectromigration :tnd by chromatography. In freshly prepared, aerated, ammoiii:ic:il solut~ions,the oxidized cobalt iuns were more adsorbed thun nickel ions and migrated at a much Yloxer rate. Sepnrntc t w t s sho\ved the osidizd c o h l t to mi-
grate slower than nickel in chromatographic system as well as in rlectrochromatographic systems. As indicated by Figure 31, arsenious and antimonous ions were readily separable by the discontinuous procedure. Under the same conditions, stannous ions could not be located in the electrographic cell with sulfide, although these ions were readiljdetectable in the cells operated continuously, as shown by Figure 16. Tests in paper strips indicated that the stannous ions were more adsorbed than arsenious and antimonous ions and that they formed a large t,railing zone. -1s a consequence, t.heir concentration in the discontinuous proccdure was reduced until they were not rcadily detect,zble. DISCUSSION
Separation of mixtlu es in the electrographic cells presents a number of advantages over the usual chromatographic systems This method not only provides a means for the continuous resolution of mistures, but it also provides a basis for the complete qeparation of many groups of ions from one another. Kith ethylenediamine tetraacetic acid as a complex-forming ~ o l u t e , this electrographic procedure makes possible the complete and rontinuous separation of monovalent cations from divalent and pol y v n l ent cations , I t RI*O provides a continuous method for the
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Continuous and Discontinuous Separations
Silver an& iiickel nitrates, each 0.005 .If in I V L , 0.005 ‘If Versene in 4 -11 NHIOH. R , diphenylthiocarbazone aud dithio-oxalic acid plus HC1. 160 volts, 95 ma. Silver and nickel separated b y electrochromatography, upper paper strip, and b y chromatography, paper strip a t right. Each ion 0.005 Jf in electrplyte and IVL,, 4 hf KHIOH. R. dithio-oxamide and diphenylthiocarbazone. Solid vector, calculated path of silver a n d nickel i n electrographic cell. Dashed vector, calcul+ted_path of silver and nickel with low electrical current and rapid How of W L Relative migration rates of silrer and nickel nitrates, each 0.005 M in W L 4 Jf NHIOH. 160 volts, ca. 60 ma., ca. 18 minutes Silver and copper separated b y electromigration, upper paper strip, and by’ohrornatography. paper strip a t right. Each ion 0.005 Af in W L , 0.01 -11 Yersene in 4 X NHdOH. R , dithio-oxamide and diphenylthiocarbazone. Solid lines, observed paths in electrographic cell Silver and nickel nitrates, each 0.005 Jf (0.01,nil.). W L ,4 M XHIOH (60 ml.). Arrow, point of addition. R , dithio-oxamide and diphenylthiocarbazone 160 volts ca 40 ma. 20 minutes Copper and nickel nitrates. each 0.005 -TI (0.01 ml.). W i , 0.01 M ammoni,um acdtate in 15 31 XHIOH (60 ml.). R. dithio-oxamide. 200 volts, ca. 35 ma., 20 minutes
ANALYTICAL CHEMISTRY
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Figures 26 to 31. 26.
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Mercurous, lead and silver nitrates, each 0.05 Jf in 1 .TI HSOa (0.01 nil,). TVL, 0.1 -71 lactic acid (60 ml.). R , HrS. 950 volts, 100 ma., 20 minutes Mercurous, lead and silrer nitrates, each 0.05 Jl in 1 M HNOa (0.01 nil.). W L , 0.008 .If citric acid in 4 :lf NHiOH (60 ml.). R, HIS. 160 to 170 volts 100 ma., 20 minutes tartaric acid (0.01 1111.). W L , 0.01 M ammonium Nickel, ferric cobalt copper cadmium and silver nitrates each'0.05 A4 in 0.1 tartrate ca. 0:005 M bimethylg1;oxime in 4 M ";OH (60 ml,). R H2S. 160 volts. 95 to 100 ma 20 minutes Mercuric bisAuth copper lead and cadmium nitrates each 0.05 M in 1'M HKOa (0.01 nil.). W L , 0 . 1 " M lactic acid (60 ml.). E , dipheAy1carbaz;de (Ag),'diphknylthiocarbazone (Cd): dithio-oxamide (Cu), dithio-oxamide plus SHaOH ( P b ) , PU'azS (BI). 250 volts, 100 ma., 20 minutes Kickel, cobalt, ferric, and aluminum nitrates, each metal 0.005 M in 0.01 M tartaric acid and 0.003 .lI dimethylgl$oxime (0.025 m1.1. U'L, 0.01 M tartaric acid and 0.005 ilf dimethylglyoxime in 4 A4 NHiOH (60 ml.), R,dithio-osaniide. aluminon in 50% acetic acid. 150 volts, 100 ma. 20 minutes Arsenious and antimonous chlorides, each 0.01 AI (0.05 ml.). Soiution and W L , 0.04 JI di-alanine in 0.1 .M lactic acid. R,HIS. 300 volts, 100 ma., 20 minutes
removal of particular cations or anions from a solution, and it makes possible the continuous substitution of one cation or anion for another, In these latter respects, it provides a promising approach to various decontamination and purification procedures. The discontinuous electrographic method provides a convenient means for the rapid resolution of mixtures and for the identification of the components. As in conventional chromatography, resolved ionic substances may be identified by their location relative to other ionic species and by radiographic tracer techniques, By analogy with separations obtained in columns, the separation of ions at tracer levels should also be possible. In the continuous procedure, separation of two ions is impossible if their relative electromigration and chromatographic rates are the same. In the discontinuous method, two ions will be inseparable only if both the electromigration and the chromatographic rates are identical. For this reason, the discontinuous method is more selective than the continuous method. In the experiments reported here, the use of organic solutes has facilitated the separation of various mixtures of cations. Conversely, various cations may now be utilized to aid in the separation of neutral organic compounds that will form complex ionic species. Similarly, the chromatographic and the electrochromatographic behavior of ions in various solvents should give indication of the formation and the nature of complex ions. Through variation of the solvent and the complexing agents and
through use of. various adsorptive agents, the electrographic method should prove fully as adaptable and as useful as the familiar chromatographic procedures. LITERATURE CITED Bailar, J. C., Jr., C h n . Rem., 21, 1 (1937). B u t l e r , J. 8 . V., a n d S t e p h e n , J. 11,L., S n t u r e , 160, 469 (1947). Clegg, D. L., A s . 4 ~ CHEY., . 22, 48 (1950). C o n s d e n , R., G o r d o n , -4.H . , a n d M a r t i n , A. J. P., Biochem. J . , 40, 33 (1946). ( 5 ) Copley, 11.J., Foster, L. R., a n d Bailar, J. C., Jr., Chem. REES., 30. 227 11942). (6) Gem :; H . - D . , a n d Tiselius, A., Biochem. Z., 220, 273 (1950). (7) D i e h l , H . , Chenz. Rtcs., 21, 39 (193i). (8) Durrum, E. L., C h e m . EILQ. Areus,27, 601 (1949); J . Am. Chem. Soc., 72,2943 (19.50). (9) G a r r i s o n , W. >I,, H a y n i o n d , H. R., a n d hlaxwell, R. D., J . Chem. Phys., 17, 665 (1949). (10) G o r d o n . A. H.. K e i l , B.. a n d Sebesta, K., Nature, 164, 498 (1949). (11) H a u g a a r d , G., a n d K r o n e r , T. D., J . Am. Chem. Soc., 70, 2135 (1948). (12) K e n d a l l , J., Science, 67, 163 (1928). (13) K r a u s , K., and Smith, G. W.,J . Am. Chem. SOC.,72, 4329 (1950). (14) Lecoq, H . , Bull. soc. m y . sci. LiBge, 13, 20 (1944). (15) L e d e r e r , E., "Progrhs R e c e n t s d e l a C h r o m a t o g r a p h i e , " Part 1, P a r i s , H e r m a n n 8: Cie., 1949. (16) Schoofs, F.,a n d L e c o q , H . , Bull. mad. T O Y . mid. Belg., 9, 122 (1944).
(1) (2) (3) (4)
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V O L U M E 23. N O . 6, J U N E 1 9 5 1 (17) S t r a i n , H. H., ANAL.CHEM.,22, 4 1 (1050). (18) S t r a i n , H.H., CarnegieImt. Wmh., Year Book, 48, 87 (1049).
H. H., “Chrornatographio Adsorption Analysis,” New York, Interscience Publishers, 1942. (20) Strain, A. H.. in “ F r o n t i e r s in Colloid Chemistry,” Vol. VIII, PP. 29-63, New York, Interscience Publishers, 1950. (21) S t r a i n , H.H., J . An. Chem. Soc., 61, 1292 (1930). (22) Svensson, H., and B r a t t s t e n , I., A ~ k i vKemi Mineral. Geol.. 1, 401 (1949). (19) S t r a i n ,
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F.,and Enenkel. H. J., Natunuissenscha/ten. 37, 93 (1950). (24) West, P. W., .~N.AT,.CHEM., 22,79 (1950). (25) Wielsnd, T.,and Fischer, E.,Natwmissenschaften, 35, 29 (1948); Angew. Chhem., 60, 313 (1940); Ann., 564, 152 (1949). ( 2 3 ) Turba,
Rrczrvno August 25, 1930. Presented at the Gordon Research Conferences. New Hampton, N. H., July 11, 1950. and before the Divisions of Physical and ~~~~~~~i~ and ~ ~ ~ 1Chemistry ~ t i at ~ the ~ 118th i Meeting of tho A a ~ n r c r aC ~ m n c ~Sro. c r m ~ Chicago, . Ill.
Techniques and Reagents for Paper Chromatography GERRIT TOENNIES AND JOSEPH J. KOLB Institute for Cancer Research and Lankenau Hospital Research Institute, Philadelphia 11, Pa.
It describes an inexpensive, transparent tank with accessories and means of wlor development on chromatographed sheets. All reagents are applied by dipping, instead of spraying; ninhydrin, in anhydrous acetone. Also desoribed are nitroprusside and cyanide dipping reagents of low water wntent, which pmduee relatively permanent colors with -SH and -S-S-, as well as a platinum and a palladium reagent for detection of reducing w m pounds in general. A n arrangement is shown for production of ineruensive chmmatoeraohic rewrds hy three-filter photography in transmitted light.
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aluminum dr glass hoo& is used to hold the iowest pair df rods apart. The papers, 40 x 46 em. in size, are marked off in centimeters on the long edges to aid in adjustment and observation of flow rate, and are held in place by glass “clothespins.” The starting line is 6 em. from the end of the sheet and should be located above tho lowest rod. The solvent is contained in 8. semicircular glass trough (51 mm. in outside diameter, 48 em. long, Yonkers Laboratory Supply Co., Yonkers 2, N. Y.). Each sheet passes from the bottom of the trough, on the inside of one of the lowest pair of rods, and turns back over the upper rod and dong the inside of the middle rod. A long filling funnel (Figure 1) with a ground inner valve near the bottom LS used to add the solvent wit.hout disturbance after t,he sheets have heen placed.
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PPLICATION of the methods of amino acid paper ohromatography ( 1 ) to specific problems has led to several improvements in general technique. THE PAPER
Chiefly as B result of difficulties in the recovery of small amounts of methionine, Schleicher and Schuell paper No. 589 Green Ribbon was selected. With certain other paper8 there is either a marked loss of methionine--e.g., Schleioher and Schuell No. 589-r a much slower movement of diffusing liquid-.g., Whatman No. 1. Three different lots of the 589 paper differed slightly in their properties, although 3 micrograms of methionine were detectable in all eases by ninhydrin after a phenol-ammonia run. The rate of flow is practically independent of the direction with reference to the water marks. APPLICATION OF SOLUTlONS
Application of amino acids or protein hydrolyeates in 50 or 100yoformic acid solutions results in small initial spots and compact spots after chromatography. In order t o apply B uniform strip of subst,ance &crossthe width of the paper for oertain types of one-dimensional work, the authors use a hard rubber comb, the teeth of which are ground down to a length of about 0.5 mm. The comb is filled from a small aluminum trough coated with paraffin. Combs of 82mm. length (100 teeth, 2.5 mm. wide) deliver 50 to 60 @I. of an aqueous 12% amino mid solution, with an error of +1 @I. Calibration was made by ooloring the solution with phenol red and determining its amount speetrophotometrically after extraction from the paper hy 0.1 N hydrochloric acid in 85% alcohol. TANK AND ACCESSORIES
The authors use a fish aquarium available in pet stores, apX 12 X 10 inches, with a combina,t,ionof ascend-
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ieopyene paint. The’upper rim af’the tank is covered with Para-
Figure .1. Aquarium Tank and Accessories Chromatography Parafilm-sheathed binders dip, comb. nluminurn trough filling funnel, and Dlartie tongs ahown outside the tank
A typical phenol run a t 20’ to 25’ C. requires approximately 20 hours. A time plot shows that there is no break in the rate of flow a t the turning paint of the p q ~ c r . Contitot between paper and glass rods also has no effect, hut contact hetween the two sheets above the liquid in the tray must beprevented. In addition to simplicity and visibility this equipment has the advantage over the ordinary descending technique of providing for automatic stoppage of flow because of the difference in height between the terminal edge of the paper and the liquid in the trough. Thus, termination of a run during the night hours causes no trouble. However, when papers are left in the tank
