Qualitative Separations by Two-way and Three-way Electrochromatography HAROLD H. STRAIN .4rgonne ;)‘ational Laboratory, Chicago, Ill. Differential electrical migration in paper moistened with one solvent followed by transverse electromigration after variation of the solvent has been tested as a qualitative analytical method for the separation of the cations of the common qualitative groups. This method, employed with various solvents and absorptive phases, has served for the separation of very small quantities of the mixtures. Its effectiveness depends upon the concentration of the ions to be resolved, the methods for the detection of the resolved ions, and the presence of various complex-
forming solutes. The results reveal that complexes formed between metal ions and various organic solutes must be in dynamic equilibrium. They demonstrate that separations based upon electrical migration are more variable than those dependent upon the flow of solvent. The seqnences of the electromigration zones may be varied so that absolute separations of many ions are possible. Information obtained by these methods is applicable to the continuous separation of ions by the combination of electromigration plus flow of solvent.
C
ROSSCAPILLARY analysis (9) or two-dimensional chromatography (%) is a widely applicable analytical tool (8). I n this two-way method a mixture of solutes, applied as a spot near one corner of a sheet of paper, is washed or developed with one solvent, and this initial, one-way development is followed by transverse irrigation with another solvent. The separations depend upon differential migration of the solutes in the polyphase system. Floiv of electrical current without flow of solvent has also been employed for the resolution of mixtures by one-way differential migration of the solutes in various polyphase chromatographic systems such as columns of moist porous adsorbents (8,12,13,16), columns of ion elchange resins (11), strips of moist filter paper (3, 4 , 7, 10, 12, 17-19), and stacks of filter paper (5). Simultaneous flow of solvent and of electrical current has been employed for tR.o-way electrochromatographic development in sheets of paper (6, 1 7 ) . These separations are knoirn as analysis by electrochromatography (6, 12, I T ) , by electromigration (11), by electrophoresis or ionophoresis (3,4, 18, 19), and by ionography (10, 12). Through the successive use of different solvents in sheets of porous media, such as sheets of cellulose or glass-fiber paper or cells of Celite, electromigration has now proved widely applicable in two-way and three-way separations. I n most experiments reported here, flow of electrical current through absorptive sheets moistened with one solvent was followed by transverse flow of electrical current in the presence of another solvent. For the three-way development, a section of the two-way separation was placed in contact with the edges of two rectangular sheets of paper that were moistened with the third solvent. Variation of pH and the addition of complex-forming reagents have proved especially effective in the electrophoretic separation of many mixtures, and these variations were usually achieved quickly without removal of the first solvent.
dilute solution of ammonia or of volatile organic acids, the current flowing a t right angles to the plane of the paper. Glass-fiber paper was obtained from the Office of Naval Research ( I ) . This paper did not contain impurities that combined with silver ions in acid solution. I n the presence of ammonia, i t had much less affinity for cupric ions than cellulose. For comparison of the migration rates of various ions, the apparatus shown in Figures 1 and 2 has proved convenient. For one-way and two-way separations, the arrangements shown in Figure 3 have been used most. I n both arrangements the paper s uares were placed on a horizontal plastic, glass, or porcelain p?ate or on a water-cooled glass tank. This paper was treated with an excess of the electrolytic solvent, and the unabsorbed liquid was removed with absorptive tissue paper (Kleenex). Formation of acidic and basic zones a t the electrodes v a s reduced by the use of weak acids or bases as the electrolyte^ ( l 7 ) , thus eliniinating the need for the electrode vessels and buffer solutions commonly employed for one-n.aj electrophoresis in paper (3, 4,7 , 10. 17-19),
-G
E
E
Figure 1. Cell for Two-way and Three-way Electromigration
METHODS
The separations by differential electrical migration which are reported here represent an amplification of previous experience in this laboratory ( 1 7 ) . They were performed with paper strips ( 2 2 , 17) or with paper squares, usua ly about 20 X 20 or 30 X 30 cm., cut from thick, soft, commercial filter paper. Much larger sheets, 55 X 55 cm. and 20 X 200 cm., are being tested. Eaton-Dikeman paper, Grade 301, 0.030 or 0.052 inch thick, or Grade 320, 0.1 inch thick, usually gave satisfactory separations. Grade 625 was less satisfactory. Some of the papers, such as Grade 625, contained impurities (probably chlorides) that migrated to the anode and that formed precipitates with silver and lead ions in 0.1 M lactic acid. This precipitate was not formed with silver ions in 4 M ammonia. The impurities in the paper were removed by electromigration in a large volume of
Above. Cross section Below. Top view platinum wire electrodes glasm panes P,paper
2,
Solutions (10 to 50 microliters) of the mixtures t o be examined (0.005 to 0.05 M ) were placed on penciled areas ofthe paper with a fine-tipped pipet, Platinum wires (8 to 20 mils) were placed along opposite sides of the paper square and were held in place by a square plastic or glass plate, which was slightly larger than the paper. This square cover rested on the wire electrodes and scarcely touched the paper itself (Figure 1). Potential (160 to 400 volts) was applied t o the electrodes for about 15 to 30 minutes for each development.
356
V O L U M E 2 4 , NO. 2, F E B R U A R Y 1 9 5 2
357
For the detection of colorless ions separated by electrophoresis, the electrical current was discontinued, the upper glass plate was removed, and reagents were applied to the paper (as gases, as sprays, or by brushing). Ions separated in the paper were often detectable by pressing the moist paper against dry paper, such as pH test paper, that had heen impregnated with the reagent.
+
migration. This was confirmed as shown by Figure 3. In the presence of ammonia plus lactic acid (Figure 2 ) , cadmium, copper, and mercury migrated faster than lead and bismuth. If, therefore, a one-way development of these five cations in dilute lactic acid were submitted to a transverse development in the presence of ammonia, cadmium and copper should be separated from lead, and mercury should be separated from bismuth. This was confirmed experimentally as indicated by Figure 3. Variation of the solvent for two-way separations has been achieved most easily by exposure of the moist paper, after oneway development, to a gas. I n the example illustrated by Figure 3, the moist paper, after one-way development, was placed on a horizontal glass rack over concentrated ammonium hydrovide in a closed vessel. Alternatively, gaseous ammonia may be passed through the vessel. The solvent has also been vaned by cutting a small rectangular section from the one-way or two-way chromatogram and by placing this section between two strips or sheets of paper moistened with the second solvent or third solvent (Figure 4). For this procedure, the one-way development may also be carried out in a paper strip ( 1 2 , l Y ) . Occasionally the papers were dried and then sprayed with the second or third solvent until moist
Figure 2. Migration of Copper Group Cations (Each about 0.005 M ) i n Paper (20 X 20 Cm.) L e f t . In 0.1 M lactic acid Right. In 4 M ammonia plus 0.1 M lactic acid
Ions located with hydrogen sulfide
The! were also detectable by placing the opposite edges of a section of the chromatogram in contact with edges of two moist sheets of the impregnated paper so that the ions could be driven into the treated paper with electrical current, the arrangement being analogous t o that shown in Figure 4. As an example, opposite edges of a section of paper containing beparate zones of nickel and copper ions (0.001 to 0.0001 M ) in tartaric acid (1.5%) plus triethylamine (2.0%) were placed in contact with the edges of two squares of paper impregnated with dithio-oxamide and moistened with the tartaric acid plus triethylamine. m'hen the electrical current A as passed through the papers for several minutes, the copper and nickel ions migrated ton-ard the anode and reacted with the dithio-ouamide, yielding very narrow, intense11 colored Tones of the dithio-oxamde reaction products.
I
I-
+I
Figure 4. One-way Migration of Iron Group Cations with 0.1 M Lactic Acid (left) and Two-way Migration after Section from One-way Separation ( l e f t ) Was Exposed to Ammonia Vapor and to Transverse Flow of Electrical Current (right) lons located with aluminon plus acetic acid, diphenylthiocarbazide, and dithio-oxamide
0 Cu
Cd
0 Osi
-0
Pb
Cd Pb Gu Bi +Hg
l
/
-
+
/
II L
I+ I
Figure 3. One-way Migration of Copper Group Cations in 0.1 M Lactic Acid (left) and One-way Separation, aa at Ideft, after Treatment with Ammonia and Transverse Development (right) Ions located with hydrogen aulfide
Paper impregnated with various reagents has facilitated the electrophoretic separat,ion of many ions. With paper impregnated withdithio-oxanlideand with 1.5%tartaric acid as solvent, cupric and silver ions were precipitated and, along with ferric ions, did not migrate, whereas cohalt and nickel ions migrated rapidly to the cathode. With 1.5% tartaric acid plus 2% triethylamine as solvent, cobalt, nickel, copper, and silver were preripitated, but ferric ions migrated rapidly t o the anode. This variation of solvente, reagents, and pH provides n variety of conditions for the separation of ionic mixtures. From the relative migration rates of the ions (Figure 2), thc suitability of varioue solvents and reagents for the two-way and three-way separations was estimated. These comparisons also provided a basis for estimation of the relative positions of the ions in the two-way and three-way eeparations. I n the presence of lactic acid, for example (Figure 21, cadmium, lead, and copper migrated a t different rates and should be partially separated rom one anothrr and from bismuth and mercury by one--ivay
In order t o follow the courbe of the sepahtions, several sheets of filter paper n-ere often employed for the initial development. Intinlate contact between the several moistened sheets was established by rolling them with a piece of glass tubing shaped like a rolling pin. The misture v a s added, and the development was then carried out as already described. After a suitable period, the uppermost sheet of paper was removed, and the progress of the separation in this paper was determined with reagents. When the ions had been carried about two thirds of the way across the paper, the solvent was varied, and the transverse development was undertaken. S H i P E OF MIGRATION ZONES
At low concentration, most solutes formed rather homogeneous migration zones with well-defined boundaries. lT7ith increasing amount and concentration of a solute, the leading boundary of the zone moved faster than the trailing boundary so that the zones lengthened rapidly as indicated by Tables I and 11. In these experiments, Eaton-Dikeman paper, Grade 301, 0.052 inch thick, 13 X 20 em., was placed on a glass plate; it was saturated with the solvent, and 25-microliter portions of the solutions of cupric nitrate were added t o separate regions as illustrated by Figure 2. rl potential of 160 volts was then applied to the electrodes held 19 cm. apart by the glass cover. After passage of the current (about 16 ma. with ammonia solutions and 20 ma. with lactic acid) for an hour, the position of the cupric ions was determined with dithio-oxamide in methanol. In either acidic or basic bolutions, the migration rates of cupric ions a t the leading boundaries increased with increasing cohcentration, whereas the migration rates of the trailing boundaries increased slightly with concentration and then decreased rapidly a t the higher concentrations.
ANALYTICAL CHEMISTRY
358 Table I. Concentration and Electromigration of Cupric Ions in 4 M -4mmonia in Paper Cupric Nitrate in 4 M Ammonia, M 0.5
n i
Migration Migration Leadin Trailing Boundary, 6111.Boundary, Cm. 2.4 0.2 2 2
i.z
1.8 1.4
0.0001 0.00001
1.8 1.2 0.8 0.7
1 0
~~
0.01 0.001
Length of Zone, Cm. 2.7
1.1 1.1 1.0
1.3 1.2
0.5
Table 11. Concentration and Electromigration of Cupric Ions in 0.1 M Lactic Acid in Paper Cupric Nitrate in Water, ,'PI
Migration Leading Boundary, Cm.
Migration Trailing Boundary, Cm.
6.0
0.6 3.5
5.0 4.4 4.2 3.8
4.4 4.3
0.5 0.1
3.J
0.01 0.001
0.0001 0.00001
Length of Zone, Cm. 6.2 3.0 1.4 0.9
0.7
4.2 3.9
Paper made from superfine glass fiber exhibited less adsorption capacity for cupric ions than paper made from cellulose fiber. I n this glass paper, the trailing portions of the chromatographic zones were shorter than in cellulose paper. The widening of the electromigration zones due t o rapid movement of the leading boundaries was similar to that observed in paper of cellulose fiber. These experiments show that separations by electrophoresis and by conventional chromatography will be most effective a t low concentration of the solutes (less than about 0.05 M for the major constituents). At these low concentrations the detection of the separated minor constituents by chemical reagents is often difficult. Among inorganic compounds, the use of radioactive tracers and the activation of resolved solutes by neutron irradiation show great promise for increasing the sensitivity of tests for the location and identification of the ions separated by differential migration methods.
0.4
SEQUENCE O F MIGRATION ZONES
The rapid migration of solutes a t high concentration sometimes interfered with the isolation of minor, slower migrating constituents of mixtures, APan example in which 4 icf ammonia Tvas employed as solvent, nickel ions a t a concentration of 1 M migrated nearly as fast as silver ions a t 0.001 ilf, whereas nickel ions a t 0.001 M migrated only about three fourths as fast as the silver or the 1 M nickel ions. I n electromigration, the differential driving force of the electrical current (18) made possible the separation of 0.001 AI silver ions from 0.1 ilf nickel ions, whereas by solvent flow in paper, the excess nickel was not absorbed and contaminated the silver unless a very long absorptive system was employed. I n order to compare chromatographic zones with the electromigration zones, 25-microliter portions of copper nitrate solutions were placed near one edge of a sheet of Eaton-Dikeman paper, the paper was placed between glass plates, and the edge nearest the spots was dipped into the solvent ( 1 7 ) . After the solvent had risen 10 or 20 cm. (10 to 30 minutes), the paper was treated with dithio-oxamide. ThBmigrations of the copper ions relative to the migration of the solvent, R, and the lengths of the zones of cupric ions, L , are summarized in Table 111. I n these experiments, the chromatographic migration and the length of the zones depended upon the amount of the cupric ions in the paper before development. With flow of ammonia especially, the zones became very long just as they did during electrophoresis, and the region of highest concentration remained near the trailing portion or the zone.
Table 111. Concentration and Chromatographic Migration of Cupric Ions Cupric Sitrate,
M
0.5 0.1 0.01 0.001 0.0001 0.00001
4 M Ammoniaa 10 Crn. 20 Cm.
0 1 24 Lactic Acid 10 Cm. 20 Cm.
R
L
R
L
R
L
R
L
1.00 0.70
10.0 7.0 1.9
1.00 0.50 0.16 0.13 0.11 0.09
20.0 10.0 3.3
1.00 0.97 0.92 0.89 0.87 0.85
4.6 3.2 2.5
1.00 0.97 0.88
8.5 7.8 6.0
0.65
3.0
0.15 0.11 0.09
1.1
0.9
2.7 2.3
1.7
1.1
0.74
4.5
.. 0.07 0.9 1.9 0.7 .. a R = distance moved by leading cupric ions + distance moved by solvent; L = length of zone of cupric ions in centimeters after solvent migrated 10 or 20 cm.
I n many of the separations by differential electromigration, the sequence of the separated ions was identical with the chromatographic sequence obtained by flow of solvent. I n the presence of various complexing agents, however, the sequences of the ions vaned greatly (17). An indication of the complexity of these effects may be gained by perusal of Tables IV and V. As in the usual chromatographic separations, the sequence of the zones formed by electrolysis may serve as a basis for the description and identification of the solutes (14). If the original location of the mixture in the polyphase system is taken as the reference point, electrolysis yields many more positions of the zones than flow of solvent-for example, with two components, two sequences of the zones may be obtained by flow of solvent (16), but six different relative positions may be obtained by electrolysis. With cupric and nickel ions and with various solutes as complex-forming reagents, all these sequences have been obtained (Tables I V and V). VARIABLE CONDITIONS
Just as in chromatography, there are two principal variables in the electrochromatographic separations. These variables are the solvent and the absorptive phase, Because the liquid phase is preferably a conducting solution, the number of solvents that may be employed is fewer than is usual with the common chromatographic methods. There are, however, almost limitless possibilities for variation of solvent by the use of solvent mixtures, by employment of various complex-forming solutes, and by variation of pH. The number of absorptive phases that may be employed in these electrochromatographic separations is also very large. Cellulose paper has been widely used because of its availability and homogeneity. I t has some disadvantages such as its capacity for the formation of complex ions with heavy metals. Glassfiber paper (1) exhibits weaker complexing properties than cellulose paper, but the glass paper is very fragile when wet. A number of two-way separations have been made in flat beds of other absorptive materials such as Celite held between glass plates, and separations have also been made in cellulose paper and in glass paper that has been impregnated with absorptive substances such as silica gel or alumina. MlGRATlON FRO\l PREClPITATES
When cupric nitrate in 4 ill ammonia was allowed to flow directly into strips of filter paper, the rate of migration wa8 much greater than that observed when 25-microliter portions of the solution in paper were washed with the fresh ammonia. For example, the R values for 0.5, 0.1, and 0.01 1Tf cupric nitrate in 4 ill ammonia were 1.00, 1.00, and 0.40, respectively.
After formation of a precipitate and under the influence of the electrical current, small quantities of the ion remaining in solution migrated rapidly, folloived by fresh material supplied by dissolution of the precipitate. Under these conditions, the precipitate remained stationary yielding a long, dilute, leading zone. These leading zones often contained so little material that they could be detected only by the most sensitive chemical reagents or
V O L U M E 24, N O . 2, F E B R U A R Y 1 9 5 2 by radioactive tracers. As an example, copper nitrate added t o a paper containing saturated anthranilic acid solution yielded a precipitate with so little copper ion in solution that flow of electrical current did not form a leading zone detectable with dithiooxamide. But in the presence of 0.1 M lactic acid and anthranilic acid, a dist'inct leading zone of copper ions was detectable with dithio-oxamide. Similarly, ferric iron plus triethanolamine plus either a- or @-alanine yielded a precipitate n-ith a long leading anionic zone detectable with hydrogen sulfide (Table V). Indeed, in many experiments in which most of the ferric ions remained near the starting position, a very dilute zone of these ions may have contaminated the other cations. These observations are analogous to results obtained by n-ashing a precipitated organic pigment in an adsorption column \%-ithfresh solvent (13,14). zones may be contaminated Tlley sho15Tthat with traces of the ions migrating from a precipitate.
359
ganic acids in the polyphase systems, appreciable but uniform heating of t,he solutions often resuked from passage of the electrical current. With dilute solutions of very strong acids, most of the heating occurred a t t,he cathode. With dilute solutions of strong bases, most of the heating occurred a t the anode. Both these effects are ascribable to the migration of the anions or cations of the electrolyte an-ay from the cathode or anode, respectively, with resultant increase in the resistance of these regions. SEPARATIONS
Silver group cations (about 0.005 M) were separated by oneLvay electrophoresis in 0.1 lactic acid, the sequence being silver, lead, mercurous, mercuric ions, with silver migrating fastest. With acetic acid or tartaric acid as solvent, separation Of mercurous from mercuric ions was not so complete as with lactic acid. Upon transverse electrolysis in the presence of ammonia plus lactic acid, the sequence was silver and mercuric ions, lead being precipitated and mercurous ions yielding mercury and mercuric ions, Bismuth ions, if present in the mixture, remained with ELECTRO-OSMOSIS AND HEATING EFFECTS the mercuric ions during electrophoresis in lactic acid, and they remained fixed as an insoluble product in the presence of amIn some experiments a small migration (electro-osmosis) of monla, With a saturated solution of versene in n-ater, silver ions solvent was observed as has already been reported during electromigrated to the cathode, mercuric and lead ions to the anode. In chromatography in columns of siliceous earth (16). This did not water saturated with trimethylamine-a,a',a''-tricarboxylic acid, the relative migration rates or the separability of the ions silver ions migrated to the cathode, lead and mercuric ions to the during t,he short time required for the separations. anode, and mercurous ions formed a very insoluble precipitate. Ammonia did not alter this effect. The action of some complexOwing to the electrical resistance of the ammonia and of the orforming reagents on the migration of silver is summarized in Table V. _____ Copper group cations were separated by one-\Yay and two-way electromigraTable IV. Relative Chromatographic and Electrochromatographic Migration tion as indicated by Figure 2. Silver, of Copper and Kiclcel Ions (Approximately 0.01 M ) in Various Solvents with a higher migration rate than any of Chromatography - Electrochromatography these cations, was separated from them Solvent Faster Slower Faster Slower rapidly and completely in either acidic Ammonia (4 31) Si Cu Xi t o cCu u tt oo or ammoniacal solutions (Table V). Lactic acid (0.1 M) S i + Cu ,, , S i to Tin group cations, stabilized in 0.2 AI (weak ads.) Triethanolamine ( 2 % ) cu Pl-i ... S i + Cu t o tartaric acid, were separated by oneTriethanolamine ( 2 % ) plus ammonia n-ay electromigration in 0.1 M lactic Cu Si S i to c u to (4 Af1.I) acid wherein stannous ions migrated to Triethanolamine (0.2 M) plus triethylamine (0.2 Cu Ni Ni t o c u to + the cathode, arsenious ions did not miTriethanolamine (0.2 M ) plus N a O H grate, and antimonious ions migrated Cu Xi S i to cu t o + (0.05%) to the anode. In 0.05 111 tartaric acid, N a O H (0.05%) Ni + Cu (ppt.) ... Ni + Cu (ppt.) N(CHrCO0H)a (satd.;) S i + Cu .,. x i + Ci to + ... in 0.05 M tartaric acid plus 0.1 Jf lactic Cu t o + acid, and in saturated trimethylamineAmmonia (4 .If) in N(CHd2OOH)a Ni (weak + Cuads.) ... Sito+ (satd.) (weak ads.) LY, a',e"-tricarboxylic acid, both stannous Triethylamine ( 2 % ) plus tartaric acid S i + Cu . . . cu t o + S i to + and antimonious ions migrated t o the ( 1 . 5 % ;acid ) , (1.5%) anode, TThile arsenious ions remained Tartaric Xi(weak + Cuads.) , . . S i to Cu t o (weak ads.) neutral. Addition of a solution of the ions in strong hydrochloric acid to a paper saturated with 0.1 hf lactic acid resulted in Table Y. Migration of Ions in Presence of Various Solvents and Complex-Forming a very slow and incomplete Reagents separation by electrolysis. Iron and aluminum group Sequence from Original Position ( : j, Solution Kegative (:) Positive cations were separated by twoway and by three-way elecSnirnonia ( 4 .Vfj trical migration. In 0.1 Jf Ammonia ( 4 -11) plus lactic acid (0.1 .TI) Lactic acid (0.1 .If) lactic acid, chromic ions miAminonia t o 4 .TI in anthranilic acid (satd.) grated rapidly t o t h e cathode; Anthranilic acid (satd.) aluminum ions migrated more Ammonia to 4 .Ifin S ( C H K O O H j 8 (satd.) Ni Co K(C€IKOOH)3 (aatd.) + Ni slowly; ferric ions did not miAmmonia (4 .W) plus L ( + j glutamic acid grate; and chromate ions iSV"\ A g , Ni + Co + Cu + Fe: L ( ~ f ~ 1 u t a macid i c (0.7%) -Ig, Ni + Co, Cu, F e : migrated rapidly to the anode, Ammonia ( 4 M ) plus triethanolainine ( 2 % ) Ag, SICu, Fe:Co so that these four ions neie Triethanolamine ( 2 % ) Ag + Xi + Cu, C o + Fe: separated by a one-way deTriethanolamine ( 2 % ) plus lactic acid Ag, Xi, Co, Co. F e : (U.1 4 1 ) velopment. Under these conTriethanolamine (0.2 M) plus triethyld i t i o n s manganous, cobalt, amine (0.2 .lf) A g , XI, F e , C u , Co Triethanolamine ( 2 4 @ )pliis m-u-alanine nickel, and zinc ions migrated /9c7-) C u : F e (pptd.), Co + Ni + - i g + Fe with the chromic ions. If the Trie&nolamine ( 2 % ) plus @-alanine( 2 % ) C o T Si + C u : F e (pptd.;), Co + A g + Fe chromic ions were oxidized to Triethylamine (2%) Ag + Xi + Co + Cu + Fe:(all pptd.) Triethylamine ( 2 % ) plus urea ( 2 % ) A g + SI+ Co + Cu + F e : (all pptd.) chromate before the one-way Triethylamine ( 2 7 , ) plus L ( + ) glutamic development in acid, a sucAg. F e : C u + Co + pii acid ( 2 % ) Triekglamine ( 2 7 @ ) plus DL-a-alanine ceeding two-way development : F e (pptd.;) C u , Co 4- Ni + AF (2%) in ammoniacal solution sepaTriethylamine (2%) plus ,%alanine ( 2 % ) : F e + Co (pptd.) Cu, Co + Ki + .4p. rated most of the elements Triethylamine ( 2 % ) plus tartaric acid (1 Ye of this group. Manganese reTartaric acid (1.5%) mained in an insoluble form; DL-histidine monohydrochloride (1mc) Potassium cyanide (0.4%;) cobalt oxidized to a cationic form and migrated slowly from
+
.J
ANALYTICAL CHEMISTRY
360 manganese; and nickel plus zinc migrated away from the cobalt ions, the nickel moving slightly faster than the zinc. With ammonia plus 2% triethanolamne as electrolyte, zinc migrated dower than nickel, and cobalt migrated slightly toward the anode. Use of this solvent for three-way development of a strip cut from the two-way chromatogram improved the separation of cobalt, manganese, zinc, and nickel. Subgroups of theiron and aluminum family were readily separable by one-way electrolysis. In addition t o the separation of ferric, aluminum, chromic, and chromate ions, ferric and manganous ions and aluminum, zinc, and chromate ions were also separated in lactic acid. Aluminum, zinc, and chromate ions were separated in lactic acid, in 2% triethanolamine plus 4 iM ammonia, and in 4 M ammonia. In triethanolamine (2%).plus triethylamine (2%) both aluminum and zinc were weakly anionic. Cobalt and nickel were separated in 4 M ammonia, in 4 M ammonia plus 2% triethanolamine, and in triethanolamine (2%) plus triethylamine (2%) (Table V). Ions of several groups exhibited the electrical migration behavior summarized in Table V. In the presence of complexforming agents, many cations exhibited anionic properties, and with different reagents, various ions of the mixtures could be made the leading electrochromatographic zones. These results indicate that various solvents may be utilized to effect a complete separation of particular components from mixtures. Thus with triethanolamine plus triethylamine, copper and cobalt may be separated completely from nickel or silver; with trimethylamine-cu,cu’,a”-tricarbo~ylic acid, copper and nickel may be separated completely from cobalt and silver; with ammonia plue triethanolamine, cobalt may be separated completely from copper, nickel, and silver; with triethanolamine plus glutamic acid, copper, cobalt, and nickel may be prepared free of silver. As each ion formed a single zone during electromigration, the complexes with organic solutee must have been in dynamic equilibnum. Mixtures of variow oiganic compounds \vere readily separated by one-way and by two-way electrolysis. In 4 Jf ammonia, pyrogallol migrated to the anode and separated from neutral glucose and sucrose, and weak bases such as triethanolamine separated rapidly from acidic substances such as anthranilic, arsanilic, sa i c y h , and sulfanilic acid. In 0.1 M acetic acid solution, anthranilic acid migrated to the cathode and separated from salicylic acid, which migrated rapidly to the anode, and from p-arsanilic acid, ahich migrated slowly to the anode. By one-way development in 0.1 M acetic acid, sulfanilic acid migrated rapidly to the anode and separated from arsanilic acid, which remained nith neutral surcrose. In succeeding two-way development in the presence of ammonia, the arsanilic acid migrated rapidly to the anode and separated from the neutral sucrov
SUMMARY
Two-way and three-way electrolytic development extend the scope of chromatographic methods. Variation of solvent, with concomitant variation of adsorbability, absorption sequence (15), and ionic mobility, facilitates the resolution of mixtures and the isolation and identification of the components. Accordingly, two-way and three-way electromigration in polyphase systems must be regarded ae extremely sensitive and widely applicable analytical tools. LITERATURE CITED
Callinan, T. D., Lucas, R. T., and Bowers, R. C., “Report of Naval Research Laboratory Progress,” pp. $18, Washington, D. C., May 1951. Consden, T., Gordon, A. H., and Martin, .1.J. P., Biochem. J . , 38, 224 (1944). Cremer, H-D., and Tiselius. .1.,Biochem. Z . , 520, 273 (1950). Durrum, E. L., ,J. A n i . Chem. S O C . , 72, 2943 (1950). Garrison, W. AI., Haymond. €1. R.. arid Maxwell, R. D., J . Chem. Phgs., 17, 665 (1949). Haugaard, G., and Kroner, T. D., J . A m , Chrvn. Soc., 70, 2135 (1948). Kraus, K., and Smith, G., Ibid., 72,4329 (1950). Lederer, E., “ProgrBs recents de la chromatographie.” Paris, Hermann et Cio, 1949. Liesegang, R. E., Natunuisser~.schui‘tan.31, 348 (1943); Z. unul. Chem., 126, 173 (1943). h‘IcDonald, H. J., Urbin, l‘f, C., and Killiamson, M . B.. Sc,ience, 112, 227 (1950). Spiegler, K. S . , and Coryell, C. D., [bid., 113, 546 (1951). Strain, H. H., A s a L . CHEM.,23, 25 (1951). Strain, H. H., in “Frontiers in Colloid Chemistry,” by R. E. Burk and 0. Grummitt, pp, 29-63, New York, Interscience Publishers, 1950. Strain, H. €I., Ind. EnQ.CI~eiia.,42, 1307 (1950). Strain, H. H., IND. ESG. CHEM.,ANAL.ED., 18, 605 (1946). Strain, H. H., .I. Am. Chem. Soc., 61, 1292 (1939). Strain, H. H., and Sullivan, J. C., . ~ N A L .CHEM.,23, 816 (1951). Turba, F., and Enenkel, H. J., Naturwissenschaften, 37, 93 (1950). Wieland, T., and Eischer, E., Ibid., 35,29 (1948); dngew. Chem., A60, 313 (1948); A n n . , 564, 152 (1949). RECEKEDApril 13, 1951.
Analysis of Mixtures of Carboxylic Acids By Spectrophotometric Determination of Rate of Reaction with Diphenyldiazomethane JOHN D. ROBERTS AND CLARE McG. REGAN Department of Chemistry and Laboratory f o r Nuclear Science and Engineering, Massachusetts Institute of Zechnology, Cambridge, Mass.
I
N THE course of investigations (3-4) of the reactions of diazo
compounds with various types of acids, the authors have had occaaion to develop methods for analysis of mixtures of carboxylic acids by measurement of their reaction rates with diphenyldiazomethane. The carboxylic acid-diphenyldiazomethane reaction has obvious advantages for this purpose, as it is very convenient to follow spectrophotometrically (the diazo compound has a deep permanganatelike color), occurs in a wide range of solvents, and may be carried out with small amounts of materials. Furthermore, different carboxylic acids often show substantial differences in rate-for example, p-nitrobenzoic acid reacts approximately 25 times faster than p-aminobenzoic acid in ethyl alcohol a t 30”. The principal disadvantages of the reaction for analytical purposes are the lack of simple stoichiometry in sol-
vents like ethyl alcohol, 15 here the reaction rate is strictly secondorder, and nonintegral kinetic orders in carboxylic acid in aprotic solvents, where the reaction is quantitative. It is the purpose of the present paper to show how these disadvantages may be overcome in the analysis of binary acid mixtures. Detailed procedures for the spectrophotometric determination of the reaction rate of diphenyldiazomethane with acids using a Beckman DU spectrophotometer with a thermostated cell compartment have been published (1-4). In ethyl alcohol, the instantaneous rate of the reaction between carboxylic acids and diphenyldiazometharle is strictly proportional to the first powers of the acid and diazo compound concentrations. However, the acid not only reacts directly to yield the corresponding benzhydryl ester but also catalyzes the forma-
