Electrochromatographic Separations of Calcium and Phosphate Ions Factors lnjluencing Separations TAKUYA R. SATO, WALTER E. ICISIELESKI, WILLIAM P. NORRIS, AND HAROLD H. STRhIN Argonne Xational Laboratory, Lemont, 111. These investigations were designed to provide simple, widely applicable, sensitive, and efficient procedures for detection, separation, purification, and estimation of calcium and phosphate ions, particularly radioactive preparations employed as tracers and radioactive species encountered i n neutron activation analyses. These procedures should be applicable to the separation of the smallest detectable quantities of tracers and t h e isolation of milligram quantities. Differential electrical migration from a narrow zone of t h e mixture in a n electrolytic solution supported in paper was found to have many desirable features. The separations were affected by t h e dimensions of the paper, the electrolytic solution, its concentration, the volume and concentration of t h e mixture to be resolved, the presence of salts and of mineral acids in this mixture, the electrical po-
S
EVERAL recent review have indicated the usefulness of
electrochromatography as an analytical tool and as an observational and diagnostic aid (8. 11, 13, 16, 21, 23-28, 31-33). These reviews have also summarized the recent applications of this method to the separation of various inorganic ions (1, 9, 11, 12, 17, 22, 27-29), ionized molecules (S-5: 7,13-15, 25, 26, SO, 34), and charged particles such as the proteins (3-7, 10, 13-16, 18, SO-SS). These applications of the methods have now stimulated the adaptation of electrochromatography to the separation of mixtures of calcium and phosphate ions and to the purification of radioactive calcium and phosphate preparations. Electrochromatography depends upon the differential electrical migration of ions from a narrow zone of the mixture in an electrolytic solvent immobilized in a porous support such as a packed column (26) or a strip or pad of filter paper (3-5, 29). It is a modification of the long-known, moving boundary method ( S l - S S ) , in which the zone of migrating ions is made very small relative to the distance of migration and in which the mixing of the solutions a t the boundaries is retarded by the porous support ($6). Dependent upon the electrolytic solution and upon the immobilization support, the migration of the ions may occur in the solution and on or in the support itself (10, 13-15, 22, 29). By analogy with conventional chromatography, which is based upon differential migration produced by flow of solvent through sorptive media (27), electrochromatography should be subject to enormous variation, particularly with respect to the apparatus, the electrolytic solvent, and the porous supporting material. Many variable features of this electromigration method have already been investigated, notably the mobilities (10, 13-16, 51-33) and the relative or comparative mobilities (3-5, 20, 21, 25, 28, 29) of the charged species, the electrode reactions, and arrangements (1, 5-5, 10, 13, 17, 20-22, 31-SS), and the nature ’ and concentration of the electrolyte and of the mixture (16, 20, 21, 29, S1-SS). Many other variable features such as the use of solutions of organic acids as electrolytes and the sorption of ions by the porous support ($1,19) have not been extensively investigated; hence effects produced by their variation are not
438
tential, the electrode reactions, and the distance of migration. The effects of variation of a few of these conditions were predictable from previous experience b u t the effects of variation of many other conditions required systematic investigation of t h e procedures. .4 simplified form of the migration apparatus with the electrodes in contact with moist paper yields reproducible migrations and separations when lactic acid of suitable concentration is the electrolyte. Sorption of calcium ions by the paper is a n important factor in t h e separations; electroosmosis is not important. Electrical migration may serve for the complete separation of calcium and phosphate ions and the separated ions may be recovered quantitatively. Effective analytical and purification procedures are being utilized i n nuclear, chemical, and biological investigations.
predictable. Moreover, many of the conditions that are requisite for the prerise determination of the mobilities of ions are not critical for the resolution of mixtures. As an example, the mobilities of ions are usunlly determined at concentrations very much lo~.~.er than those of the supporting electrolyte (16, 51-53), but practical separations may be obtained even Tvhen the concentration of the mixture approaches that of the electrolyte (25,28,29) Because of the need for simple, convenient, analytical procedures for the separation of calcium and phosphate ions, various qualitative and quantitative aspects of the electrochromatographic separations have now been explored. The effect of variation of the migration systems has been examined; the effect of the nature and the concentration of the supporting electrolyte upon the migration of the zones has been investigated; and the effect of the concentration of the migrating solutes upon the size and the shape of the zones has been tested. The effects of the volume and the concentration of the mixture upon zone formation, upon zone boundaries, and upon separability have been established. The effect of the sorption capacity of the porous phase upon the mobility of the solutes has been examined by the use of the common chromatographic procedure. The role of electro-osmosis in the separations has been investigated with water containing tritium as the indicator. This information, which extends many earlier investigations, has provided a practical basis for the rapid qualitative and quantitative separations of mixtures of calcium and phosphate ions and for the purification of these ions. It has revealed many unsuspected phenomena such as the role of sorption on the mobilities of the ions and the effect of various conditions upon the distribution of the ions in the migrating zones. MATERIALS AYD APPARATUS
Radioactive, “carrier-free” calcium and phosphate ions were obtained from Oak Ridge Xational Laboratory. Some of these preparations of high specific radioactivity contained active impurities that were removed by electrical migration, as described in a subsequent section (Figures 16 and 17).
439
V O L U M E 25, NO. 3, M A R C H 1 9 5 3 Most separations reported here were performed in paper moistened with a single supporting electrolyte, aqueous lactic acid. Lactic mid solutions are suitable eonduetors of the electrical current, they undergo slow changes in concentration a t t,he electrodes, and they dissolve many ions (to, 81, 26, t8, 29). Several filter papers, including glass-fiber paper, were tested as pqrous qedia for the resolution of mixtures by differential electrical mwation. Because of its thickness, toughness, and uniformity, -a commercial paper of wood pulp (Eaton-Dikeman, Grade 301, 0.03 inch thick) has been employed in all the experiments described herein. It is available in rolls 6 feet wide. It is exceptionally permeable to aqueous solutions; hence it is useful for the formation of chromatograms which may be developed quickly by flow of solvent. Far the control of the temperature of the moist paper during the electrical mieration, a flat glass cell (a Thermapsne window 3
the plastic sheet, and'the lac& acid salution-w& added until an excess was present in the paper. After a. few minutes, the excem electrolytic solvent x m s removed with blotting paper (Kleenex). Solutions of mixtures t o be investigated were then added from sharp-pointed micropipets t o penciled circles on the paper. I'littinum wire electrodes were placed along opposite ends of the paper (26),which was then covered loosely from both sides with the polyethylene sheet. This sheet was sealed together with a little silicone grease; the platinum wire eleotrodcs were pressed firmly to the paper between plastic (Lucite) strips held with smew clamps; and potential (200 to 500 volts) from an electronic rectifier ( $ 1 ) was applied t o the electrodes. Measurements with thermocouples showed that the temperature of the paper did not rise more than 2' above the temperature of the cooling water, which varied seasonally from about 8" to 18". The Thermopane cells were susceptible t o shock caused by sudden change in the water pressure. They were, therefore, protected by w e of a pressure regulator delivering the feed water with about %em. head. These cells were also protected from mechanioal strain by a heavy plywood support. Heavy objects and clamps were not placed on the Thermopane. DETECTION AND IDENTIFICATION O F SEPARATED IONS
The migration of radioactive ions in the moist paper was followed with a thin-window Geiger-Miiller counter. When the
These autographs revealed the distribution of the acdvity within the eones. and they provided a permanent record of the separa
'5 hours a t 5'volts per om., the radioactivezones were readily detectable after an exposure of some 12 hours. Concentrations ranging from 0.05 to 10 pc. per 50 PI. yielded uniform zones that migrated a t the same rate when electrolyzed side by side in a single sheet of paper (30 X 80 om.). Radioactive ions separated in the filter paper were sometimes estimated directly with a calibrated Geiger-Muller tube and scaler. They were also estimated after extraction from the paper by the elution techniques employed in conventional chromatography (Figure 1)
Figure 2. Variation ofConductance of Moist Paper Strips G i t h Time Parallel strips (5 x 80 om.) moistened with lactio acid of various conoentratlon. Electrodes elamped t o paper. Potential, 400 volts per 80 cm.
Nonradioactive ions and acidic and basic zones in the paper were located with various reagents and with acid-base indicators. The sensitivity of the best reagents never approached that achieved by the use of radioactive tracers. CONDUCTANCE IN PAPER MOISTENED WITH LACTIC ACID
raombr hdod; and ihe separated ions were located photographically (Kodak no-screen x-ray film, 14 X 17 inches; dark room).
Conductance (direct current in milliamperes) in paper moistened with lactic acid, employed as a test of the stability of the migration system, varied with the acid concentration and with the method of moistening the paper. In one series of tests, the paper strips ( 5 X 50 cm.) were moistened with the lactic acid and blotted with Kleenex; in another series, the strips were washed for 1, 2, or 3 days by continuous downward percolation. With aoid of concentration less than 0.001 M , the conductance of the washed strips was slightly less than that of the blotted strips. With acid more concentrated than 0.01 M , the conductance of the washed strips was greater than that of the blotted strips. Continued eleotrolysis of the migration system produced a slow decrease of the conductance (Figure 2). VARIATION OF CONCENTRATION AND OF POTENTIAL GRADIENT DURING ELECTROLYSIS
Figure 1. Apparatus for Development of C h r o m a t o g r a m s and Elution of Separated Substances Height of tank for solvent adjustable by knob, screw, and levers. Plastic ooyer &tleft
To locate the regions in whioh electrolysis altered the paperlactic acid system, a potential of 400 volts was applied to moistened paper StripS(5 X 80 em.) and the potential difference hetween successive lO-cm. regions was determined. At the higher concentrations of lactic acid, there was a remarkably uniform potential gradient in all sections of the paper, except in the 10em. region in contact with the cathode. I n this region and with 1.0 M acid, the potential difference rose from an initial 50 volts to 65 and 70 volts afte? 25 and 50 hours, respectively. The corresponding rise for 0.5 M acid was 65 and 85 volts, and for 0.1 M acid, 150 and 205 volts. With these increases there wae a
ANALYTICAL CHEMISTRY
440
decrease bf some 10 to 20 volts in the other 10-cm. regions of the paper. With acid of concentrations .If or less, the zone of high resistancemoved rapidly from the cathode, appearing in the first 10 cm. of paper in ahout 5 hours, in the second 10 cm. in 7 to 20 hours, and in the t,hird 10 cm. in 10 t.o 40 hours. The initial inarease in potential gradient a t the cathode was followed by a rapid decrease and by an increase in pH to as much as 11 to 12. The initial increase of the potential gradient at the cathode must be ascribed to a depletion of lactate ions, the subsequent decrease to an accumulation of c:itians present as impurities in the solution and in the paper. For the resolution of mixtures (particularly with the electrodes in contact with the paper), the lactic acid concentration should be about 0.1 M or greater. With a potential of 5 to 10 volts per em. the electrolysis should not be carried out for more than 10 to 2o hours. Themination s?stem be long enough that the migrating mixtures traverse only t,he central region of the
..r-_.
llrl-l-.l..
-2
~ - ~ ~ " ~ e ~ o f P " ; i f ~ ~ - ~ ~ i ~ ~ ~ ~ - g ; ;in; lo,l -Phosp Lactic Acid
Eaoh none aonfained 50 pl. of 0.5 M HCI vi% 0.3 PD. of each tracer plus rariDUB anruiints of CaHPOt: 1 0.5 iM. 2 0.1 M : 3 0.01 M . 4 0.001 M . 5 0.0001 M . Potential, 400 vblts oer'8O'cm. Mi&tion timh, d hours. $a16 shown in figure. Ca sones at left
STARTING POINT
Figure 5. Effect of Concentration upon Separation of Mixtures of Purified Calcium and Phosphate Ions in IO-& M Lactic Acid Emh zone oontained 50 d. of 0.5 M HCI with 0.3 w , of eaah tracer plus variorir concentrations of CaHPO,: A , 0.5 M; E , 0.1 M: C 0.01 M . D 0.001 M: E . o.ooo1 M . Potential. 400 per 80 Cm_ ~ & , i i Lime: ~ ~ B hours. A to E , 23 cm. Calcium zone E , 1.5 cm. long. Phosphorus sone E . 3.0 om. long st rizht
paper. Far the precise determination of ionic mobilities in very dilute lactio acid or in dilute salt solutions, however, the ends of the paper and the electrodes should he placed in lssge volume of the electrolytic solutions as commonly practiced (Sd, 10, 16, S1-SS). AMOUNT OF MIXTURE AND ZONE FORMATION IN PAPER MOISTENED WlTH LACTIC ACfD
Figure 3. Effect of Volume and Activity of Solution upon Separation of Mixtures of Purified, Carrier-Free Cad6 and
PWd Ions A.
B.
C.
D. E.
F.
5 PI., each ion 1 w . 15 PI., each !on 3 w. 25 MI., each m n 5 PD. 50 d.,each ion 10 PO. 100 PI. each ion 20 pe. 200 PI.: each ion 40 PD.
fs,s& :E,
To prevent excessive enlssgement of the migrating eones, not more than about 50 wl. of the mixture was added to the moist paper (Figure 3). This provided an initial zone about 1 em. in diameter and final zones several centimeters in diameter. As expected, the concentration of the solution of mixture? added to paper-lactic acid affected the size, the shape, and the migration behavior of the zones. But unexpectedly, these effects did not become troublesome until the concentration of the mixture approached that of the lactic acid (Figures 4 and 5 ) . Similar results were also obtained with calcium, cupric, and phos-
&~~~~~~~
~ , ~ h $ & ~ ~ ~ i ~ , o L ~ ~ ~ ~ $ p ~ ~ > m ; h ~ , & ' ~ 6.5 cm. long. Before photographie reduction of radioautograph, distance between origin and phowhste zone8 was shortened by 6.5 am.: and distame between origin and chloium zones w86 shortened by 12.6 cm.
V O L U M E 25, NO. 3, M A R C H 1 9 5 3 STARTING POINT
Figure 6. Effect of Hydrochloric Acid upon Separation of Calcium and Phosphate Ions i n 0.1 M Lactic Acid
phrtte ions added separately (and without hydrochloric aeid) to the moist paper. MINERAL ACIDS AND SEPARATION OF CALCIUM AND PHOSPHATE IONS
As cdcium phosphate is sparingly soluble in water, mineral acids were added to the mixtures of calcium and phosphate ions to be separated by e l e c t r o m i g r a t i o n in paper plus lactic acid Surprisingly. hydrochloric acid a t concentrations of 0.5 A l h a d little effect upon the migration of the zones of calcium and phosphate ions (Figures 4 and 5 ) . At concentrations of about 5 M , however, both nitric and hydroohlorio acids and mixtures of these acids retarded the migration of thezones, so that the separation of calcium and phosphate ions was slow and incomplete. At t h e cathode side of the migrating calcium zone, calcium ions migrated slowly from the POTENTIAL VOLTSlcm mineral acid into the lactic acid s o l u t i o n . Figure 7. Migration of Calcium Ions dtming 1.8 Hours in Lactic There, removed from Acid of Various Concentrations the strong m i n e r a l In 5 X 80 cm. paper strips and at v&riou~ acid, they migrated potentiak. Migrations carried ont sirnulmore rapidly toward tsoeously st eaoh potential
441
the electrode (Figure 5), forming a diffuse zone reminiscent of the diffuse leading eone observed whrn ions migrate from a precipitate (8.5, 84). I n the presence of strong nitric or hydrochloric acid (3 to 6 M ) the phosphate ions formed small, well-defined, often erescene shaped zones that migrated slower than the zones of calcium ions. Phosphoric acid a t much lower concentrations (0.1 to 0.5 M ) had little effect upon the migration of calcium ions, but it retarded the migration and produced marked elongation of the zones of radioactive phosphate. The s l o ~migration of the calcium ions in the presence of mineral acids is due to the competition with the other ions, particularly the mobile hydrogen ions. The very slaw migration of the phosphate may he due to repression of the dissociation of the phosphoric acid as well as to competition with the other ions. The exnlanation for the diffuse leading region of the oalcium zone lies in the constituent mqbilities of the various ions and in the geometry of the system. In the region of high ionic strength, the calcium migrates slowly, but as it leaves this zone, it migrates much faster. The higher the concentration of mineral acid and the larger the initial zone, the greater the time required for the separation of the calcium and the longer and the more diffuse the leading calcium zone. W i t h p h o s p h o r i c acid, on the contrar,v, the nitrate and chloride anions migrate the faster, leaving the phosphoric acid behind. 5 IO 15 20 But in the region of lower POTENTIAL VOLTS/cm ionic concentration, thie Figure 8. Migration of phosphoric acid migrates Phosphate Ions during 4.0 faster than the zone of Hours i n Lactic Acid of Various Concentrations mineral acid and remains a a distorted zone a t the In 5 x 80 ern: gaper strips and &t varrous potentiah. Migrations car. trailing boundary of the ried out simultaneously at eaoh potential acid eone. MIGRATION AS 4 FUNCTION OF WTENTIAL
In agreement with most investigations of ionic migration (10, 1s-16, S l d S ) , the mobilities of calcium and phosphate ions were directly proportional to the potential gradient, provided the gradient did not exceed some 15 volts per em. At greater gradients, however, the migration varied unpredictably, especially in lactic acid of different concentrations (Figures 7 and 8 ) . In the case of phosphate ions, and with a potential gradient of 5 volts per cm., the migration was greatest in lactic acid of lam concentration (ca. 10-6 M ) . With calcium ions, on the contrary, S in lactic acid of high concentration the migration N ~ greatest (0.1 to 0.25 M )(Figure 9). All the measurements summarized in Figure 9 represent short migration periods (2 hours); consequently, the differences among the migration rates in lactic acid of various concentrations cannot he attributed to changes in the conductance during the migration (Figure 2). Moreover, separate experiments with calcium and phosphate ions in 0.1 M lactic acid showed t h t a t 5 volts per em. the migration could he continued for 20 t o 25 hours before the migration rates decreased.
'
ANALYTICAL CHEMISTRY
442
The observed differences among the migration rates (Figure 9) may depend upon many Conditions, such as the sorption of the ions by the paper and the migration of the water as in electroosmosis (86). The roles of these two phenomena in the electrical migration of calcium and phosphate ions were, therefore, examined by electrical migration experiments with water containing tritium and by conventional chromatographic experiments with radioactive tracers. MIGRATION OF WATER CONTAINING TRITIUM IN PAPER PLUS LACTIC ACID
Far determination of the possible role of eleetro-osmosis during the migration of phosphate and calcium ions, water Iaheled with tritium w&s added (as a small zone about 1 cm. in diameter) to the paper strips containing lactic acid of various concentration
I I I
I
I. .5 2 5 .I
a1
163
,64
165
104
I
CONCENTRATION OF LACTIC ACID Figure 9. Comparison of Migration of Calcium and Phosphate lons i n Lactic Acid of Different Molal Concentrations i n 5 X 50 Cm. Paper
Table 1. Flow in Paper of Solutions of Lactic Acid and Migvation oCCalciurn and Phosphate Ions and of Tritiated Water [Relative to flow of wash liquid ( R d u e ) ( 8 ) during 1.5 hours]
M
L ~ o t i eAcid
0.5
0.1 0.01 0.001 0.0001 0.00001
Cm. 27.2
28.7 28.3 27.0
Ca'l
R
P190.
Hi0
1.0 1.0
1.0 1.0 1.0 1.0
R 1.0
1.0 0.80
28.5
0.45 0.15 0.10
28.5
C.10
1.0
1.0 1.0
1.0
1.0
CHROMATOGRAPHIC BEHAVIOR OF TRITIUM-CONTAINING WATER
In the electrolysis experiments, the slow migration of the tritium could not he attributed to sorption by the paper. This was shown by adding the labeled water t o dry paper followed by washing the paper with water, as in the development of a chromatogram (Figure 1). Under these conditions, most of the tritium migrated with the leading boundary of the water, only a small fraction forming a diffuse trailing zone. Similar effects were obtained with paper that had been washed with lactic acid of various concentrations and dried. In these chromatographic tests, the diffuse trailing boundary of the tritium zone must he attributed to the slow and incomplete reversal of the exchange betweon the tritium of the water and the hydrogen atoms of the paper. This effectshows that a migrat ing zone may leave small trailing portions to contaminate the solutes that form more slowly migrating aones (a?). CHROMATOGRAPHIC BEHAVIOR OF CALCIUM AND PHOSPHATE IONS
The affinity between paper and carrier-free calcium and phosphate ions was estimated by washing narrow zones of the tracer
The electramigration was carried out with the electrodes on the paper. The paper w&s dried in air and a t room temperature, and the residual tritium was located photographically with an exposure of several day8. I n control experiments without electrolysis, the zone of residual tritium tracer was about 3 cm. in diameter. The behavior of the zone containing the tritium during electrolysis varied with the concentration of the lactic acid (Figure 10). With acid of concentration greater than 0.1 M , there wm no enlargement and no migration of the tritium zone. With more dilute solutions, the boundary of the radioactive zone facing the anode remaihed fixed in the paper, whereas the boundary of the eone facing the cathode became diffuse and migrated toward the cathode, resulting in a lengthening of the zone itself (Figure
A
IO). Another aeries of electrolysis experiments with 0.1 M lactic acid (5 volts per cm.) showed that there was no movement of the principal tritium zone after 1, 2.5, 4,and 7.5 hours. In a third series with the ends of the paper dipping into electrode vessels containing 0.5 M lactic acid and with a potential of 5 volts per em., there was also nomigration of the water containing tritium. At lower concentrations of lactic acid, however, there was Some migration toward the cathode. All these observations indicate that migration of the lactic acid solution does not account for the difference between the migration rates of calcium and phosphate ions (Figure 9). Incidentally, these observations also throw Some light on the mechanism of the migration of hydrogen ions. As the tritium ions do not migrate rapidly toward the cathode, the meohanism of hydrogen ion transport must include interchange of these ions with d l the labile hydrogen atoms of the electrolytic system. Under these circumstances, particular hydrogen ion8 must persist for very short periods, so that they migrate a very short distance during the electrolysis.
R
.-
Figure 10. Autograph after Eleevolysis of Laotic Acid Containing Zones of Water with H:O, 50 &I.
V O L U M E 25, NO. 3, M A R C H 1 9 5 3
443
ions in paper with lactic acid solutions. In untreated paper and i n paper saturated with lactic acid and dried, phosphate ions migrated as fast as the developer solutions of lactic acid, thus showing that the phosphate ions mere not attracted by the paper. The migration of calcium ions varied with the concentration of the lactic acid used as the developer solution, indicating that thrse ions were sorbed by the paper in the presence of dilute lactic acid. When calcium ions in dry paper were washed with solutions of lactic acid, the acid itself was sorbed by the paper. In these systems, the lactic acid was located with an indicator (neut r d red, pH range G.8 to 8.0)) and the calcium zones were located by radioautographs. Comparison of the migration of the ions relative to the migration of the water ( R values) reveals that the zone of calcium ions appeared near the leading boundary of the lactic acid, especially a t the higher concentrations of lactic acid (Tables I and 11). Calcium ions were also retained in the paper previously saturated with dilute lactic acid and dried. These results shorn that calcium ions are strongly Eorbed on paper from solution in water and from solution in dilute lactic acid. They are not sorbed from lactic acid a t concentration greater than about 0.1 M .
Table 11. Variation of R \-slue (2) of Lactic Acid (Observed b y flow of acid of various concentrations through paper for hours)
Jf 0 5 0 1 0 01 0 001 0.0001 0 00001
-
Lactic Acid Solution Cm. 37 0 39 0
Lactic Acid, R 0.95 0.82 0.42
37 1
35 8 Acid zone not detectable Acid zone not detectable
0.06
No* No+ No' No' No+ No' No' No'*
-
t
GI- GI- GI- B r - B r - B r - G I - GI--
--- HL
H
H
H
H
H -
L
L
L
L
L -
+
Figure 12. Schematic Mechanism for llligration of an Acidic Zone (Circle) to Cathode with Sodium Chloride as Supporting Electrolj-te
-+ H
H
H
H
H
H
H
H
P
-L
L
L
L
L
L
L
L
GI- G I - C I -
F
I
H
H -
L
L -
-
H
H
H
H
Not No'Na'H
-L
L
L
L
L
CI-CI-GI-
-L
L p -
L-
L
P
H
L
H
H
H
H-
L
L
L
L-
t
+
Figure 11. Schematic Jlechanisni for Migration of an Acidic Zone (Circle) to Anode with Lactic Acid (HL) as Supporting Electrolj te Hydrogen ions in lower circle are different from those in upper circle.
At the higher concentrations of lactic acid, the electrical migration properties of calcium and phosphate ions (Figure 9) cannot be influenced by sorption on the paper. At the low concentrations, however, the decreased migration rate of calcium must be ascribed largely to absorption of calcium ions by the paper. The cause for the decreased migration of phosphate a t very low concentration of lactic acid t o lo-' M) is not apparent. The sorption of calcium and lactate ions by the paper and the nonsorption of phosphate ions illustrate the complexity of the sorption phenomenon and indicate an even more complex relationship among sorption phenomena, ionic migration, and electroosmosis. Incidentally, the chromatographic observations (Table I) indicate that mixtures of calcium and phosphate ions should be separable by sorption in paper strips followed by development with water and that they should not be separable by development with 0.5 M lactic acid, from which they are not sorbed. This was confirmed by chromatography of tracer quantities of calcium and phosphate ions. ELECTRIC4L IIIGRATION OF ACIDIC AND BASIC ZORES
When a few microliters of mineral acid (0.1 M) were added near the center of paper strips moistened with lactic acid, subsequent electrolysis caused the strongly acid zones to migrate toward the anode. These strongly acidic zones were readily located by spraying the paper with a solution of methyl yellow (p-dimethylaminoazobenzene, pH range 2.9 to 4.0). Even though the acidic zones migrated toward the anode, the hydrogen ions in the initial zone probably did not migrate, as indicated by the
L
L
u GI
GI
GI
L
L-
Figure 13. Schematic Mechanism for Formation of Acidic (Circle) and Basic Zones (Double Circle) by Electrolysis of Salts in 0.1 M Lactic Acid
experiments with water containing tritium. -1schematic mechanism for this migration is shown in Figure 11. This migration of the acidic zone toward the anode was observed with supporting electrolytes such as solutions of various organic acids and ammonium salts of tartaric and oxalic acids. But with solutions of lithium, sodium, and potassium chlorides (0.01 J f ) as the supporting electrolyte, small initial zones of mineral acid anions migrated to the anode, and the zones of elcess hydrogen ions migrated to the cathode (Figure 12). When narrow zones of neutral salts were electrolyzed in the paper plus lactic acid, the anions yielded acidic zones that migrated to%-ard the anode, and the cations yielded weakly basic zones that migrated toward the cathode (Figure 13). When these neutral salts were electrolyzed in paper impregnated with 0.5 M ammonium hydroxide, very weakly acidic zones migrated to the anode, and strongly basic zones migrated to the cathode (indicator; alizarin yellow R, sodium p-nitrobenzeneazosalicylate. pH range 10.1 to 12.0). Here the hydroxyl ion zone accompanied the cations, the hydroxyl ions themselves actually migrating to the anode, the inverse of Figure 11. When salts of strong acids and strong bases were electrolyzed as narrow zones in paper impregnated with sodium chloride solution, the acidic and basic zones were not formed in the renter of the paper. The formation and migration of these acidic and basic zones further demonstrate the complex and variable properties of the electrical migration system. Under some circumstances, as with the separation of proteins and of heavy metals, the forma-
444
ANALYTICAL CHEMISTRY
tion of these acidic and hasic zona may produce troublesome secondary reactionsof the migrating ions.
two znnes of migrating ions, a n indication .that most of the ions remained in their respective zones and that they were separated completely from each other (Figure 14).
COADITIONS REQUIRED FOR QUALITATIVE SEP.4R.4TIONS OF CALCIUM AND PHOSPHATE IONS
For the efficient separation of mixtures of calcium and p k o s p h t e ions and with the apparatus and paper employed here, the concentration of the lactic acid should be 0.1 to 0.5 M (Figures 7 to 9), and the ooncentration of calcium and phosphate should not exceed one tenth of this (Figures 4 and 5 ) . For the convenient detection of the separated zones, the radioactivity of the tracers in the mixture should be ahout 0.1 I",:. or greater per 50 rl. of the solution. The volume of the solut,ion added to the paper should not. exceed about 50 rl. The conrentrat.ion of nitric or hydrochloric acid added to mixtures of calcium and photphate ions should not exceed 0.5 to 1.0M . At an electrical potential of about 5 to 10 volts per om., the migrations should not he carried out far more t,han about 20 to 25 hours. STARTING
POINT
COMPLETENESS O F SEPARATIONS OF CALCIUM AND PHOSPHATE
The separation of radioactive calcium and phosphate ions was followed quantitatively hy counting the act,ivity of the separated ions.
I
ti
e
Figure 1.5. Pads of Filter Paper for separation of Calcium and Phosphate Ions
c
In I u~1.of0.1 iM solution (%OD). assmlhlcd cleetrolyrcd. and aatograubed, a n d sewrated with autograph o< initial ,.one (hott.olr)
4liquuot portions (50 p l . ) of the mixtures of the radioactive ions \\-ere added to the paper plus lactic acid and separated by elec-
c
.
Figure 14.. 3eparatlon et KadioactiYe Lalerom ana l'hosphate Ions with Absence of Radioactive Xaterial between Zones
Qualitative tests showed that calcium and phosphate ions could he separated over great ranges of conrentration. Even with radioactive tracers, the lower limit for separability wa8 the limit of deteotability (about 0.1 p c . or 3.5 X gram of phosphorus-32, assuming the "carrier-free'' phosphate to he uncontaminated with phosphorus-31). The upper limit of concentration, determined hy adding inactive carrier, depended upon the conoentration of the lactic acid and was ahout 0.01 M for 0.1 M acid. Under conditions favorable for migration, the calcium and phosphate ions yielded well-defined zones. Prolonged exposure (48 houn) of the paper t o the x-ray film did not yield detectable amounts of radioactivity in the portion of the psper between the
trolysis (Figure 14). The paper vas dried; the eones of calcium and phosphate ions %-ere cut out, and the radioactivity w m estimated in a calibrated counter. These values were compared with those of aliquot portions of calcium and of phosphate ions that had been added to pieoes of dry paper and measured in the counter under similar conditions. The recovery for calcium-45 was 98.2 and for PB*06 97.8(fl)% of the amount added to the paper. Calcium and phosphate ions separated by eleotrochromatography were d m estimated after elution from the paper. To this end, the separated ions WWP elnted hy percolation with nitric acid (1 M) (Figure 1). The eluted ions were diluted with carrier, the calcium was precipitated with oxalate, the phosphate wa8 precipitated s-ith ammonium molybdate, and the radioactivity of the precipitates w m determined. The recovery of the resolved radioactive ions was Caa5100 and Pa20498.5(+1)% of the amount added t o the paper. Calcium ions from 50 PI. of an acid solution about 0.01 M with respect to calcium phosphate were separated by electromigration in 0.1 M lactic acid. They were isolated by elution with nitric
p q e r j , ~32.8';. Recovery, 98.2% LARGE SCALE SEPARATIONS
The eleetromigration procedure %-as modified so that enlcium and phosphate ions eontsined in seveul milliliters of 0.1 Af solution may be separated. .
For this purpose, two thick pads of paper each 2.5 X 10 X 50 em. were prepared as shown in Figure 15. The mixture, containing calcium and phosphate tracer ( 4 m i ) , was placed in a narrow strip of dry paper, 0.3 X 2.5 X 10 em., that was then inserted between the two long pads previously moistened with 0.1 M lactic acid, Upon the application nf electrical potential to this system, the calcium and phosphate ions migrated as two wide. zones (Fi'jgure 15). Indications that the ions had been separated
V O L U M E 25, N O . 3, M A R C H 1 9 5 3
445
completely was obtained h r exvosure of the small uamr striD to a
by evaporatibn, and t h e calcium and phosphatk w&pr&ipit.ated oxalate and phosphomolybdate, respectively. In a typical experiment, 91.5% of the cnlcium rtnd 92.5% of the phosphate were recovered. a8
ABSOLUTE SEPARATIONS
In addition to mechanistic considerations (df,27), two lines of investigation indicated that the separation of calcium and phosphate ions by electrolysis should be 100% or absolute. Qualitative radioautographs shoused the radioactive calcium and phosphate ions were separated into discrete zones, and quantitative determinations revealed that, within the limits of error, all the calcium and ph0sphat.e ions v e r e reeovemble from the respeetive zones. Quantitative evidence for the completeness of the separation of calcium rtnd phosphate by electromigration must be based upon determination of the ertlcinm and phosphate ions a t great differences in oonoentration. This has now hoeti achieved hy the use of purified tracer dementa (aee next section) st high levels of activity. With these highly sotive solutions in paper plus lactic acid, all the phosphate ions formed a singlo zone nithout detectable amounts of radioactivity in other portions of the paper. Similarly, the radioactive calcium ions formed a single zone containing all the detectable activity. Mixtures of the two ions separated without detectable quantities of activity in the intervening portions of the paper. In these experiments, the activity of the calcium was 300 pc. and that of the phosphate was 250 pc. Because the photographic method permitted the detect,ian of traces of mdioaotivity (0.05 pc. or less per sq. em.), this experiment shorn that not more than 1 part of calcium or phosphate in 3000 rem;Lined behind the principal zones and that the intercontamination of the phosphate and calcium zones oould not have exceeded one part in several thousand. SEPARATION O F IMPURITIES FROM Cali AND
PI%
radioactive zone8 of impurities were found in the paper (Figures 16 and 17). Once these impurities had been removed by eketrioal migration, the purified ions, recovered by elution from the principal zones, yielded a single zone when re-examined by elertromigration. The radioactive impurities have not yet been identified. Those from calcium (Figure 16) emitted B-, y-, and x-rays. They amounted to about 0.1%of the total calcium activity. The trailing sones isolated from phosphate (Figure 17) were various unidentified ionic species of phosphorut-containing ions. This WRS ascertained by determination of the half-life and the intensity of the radiation. The ions from the principal zone were identical with orthophosphate, a6 shown by identical compasative electromigrstion in lsct,io acid and by precipitation with ammonium molybdst,e. STARTING POINT
I
IONS
When the radioactive ions of high activity were first submitted to electrolysis in pnper pius lactic acid, several disbinct, minor,
Figure 17. Impurities Separated as Trailing Zones by Electrical Migration of P320,Ions of High Radioactivity
STfi+.LING
T
The trailing %onesfrom the phosphate preparations sometimes contained nearly 20% of the t,otal activity. The stability of these phosphorus-containing solutes was tested in several waytys. The eluted substances were recovered by evaporation of the solutions a t 20", and they were then resubmitted to electrical migrs, tion in paper. Each solute formed II single zone identical with that obtained from the miiture. When the solutions from each zone were heated with nitric acid plus hydrogen peroxide and then resubmitted to electrical migration, the orthophosphate was unaltered; the phosphorus compounds from the other zones were converted slowly into Orthophosphate. As the radioactive phosphate is usually prepared by neutron irradiation of sulfur followed by acid oxidation and chemical separation, the trailing phosphorus-containing zones could be phosphorous compounds, various polyphosph&,etes, or sulfatophasphates. The detection of these impurities in radioactive phosphate by means of conventional chromatography has recently been reported (19, 35). Figure l b . Impurities Separated as 'Trailing Zones by Electrical Migration of Calcium Ions of Eigh Radioactivity
CONTINUOUS SEPARATIONS BY ELECTROCHROMAT(
In the method far the continuous resolution of mixvucvn UJ electromigration plus chromatography (S,6, 8, $0, 21, 29, SO) the separations depend upon the differential electrical displacement of a oontinuous, narrow zone in an electrical field. The various
ANALYTICAL CHEMISTRY
446
conditions such as concentration of electrolyte, concentration of mixture, and electrical potential now found to be critical for the one-way migration should also be determinative for the continuous method (29).
McDonald, H. J., Urbin, hI. C., and TTilliamson, hl. B., Science,
ACKNOWLEDGMENT
Miohl, H., Monatsh., 82, 489 (1951). Rouser, G., and Neuman, 14’. F., Fed. Proc., 1 1 , S o . 1, Part 1, 278 (Ilarch 1952). Sato, T. R., Norris, TV. P., and Strain, H. H., A 4 CHEiv., ~ ~ ~24,.
112, 227 (1950).
MacInnes, D. A., and Longsworth, L. G., Chem. Recs., 11, 171 (1932).
Madorsky, S. L., and Strauss, S., J . Research S a t l . B u r . Standards, 4 1 , 41 (1948); RP1901.
The authors are indebted to Jane K. Glaser for preparation of numerous slides and photographs, to Blanche Laivrence for determination of calcium, and to Robert L. Ferguson for assistance in the determination of the electrical conductance of paper strips moistened with lactic acid.
776 (1952).
Sato, T. R., Korris, W. P., and Strain, H. H., U. 8.Atomic Energy Commission, ANL-4724 (November 1951). Spiegler, K. S., and Coryell, C. D., J . Phys. Chem., 56, 106 (1952).
Strain, H. H., AYAL.CHEM.,22, 41 (1950).
LITERATURE CITED
Brewer, 8.K., hladorsky, S. L., Taylor, J. K., Dibeler, V. H., Brodt, P., Parham, 0. L., Britten, R. J., and Reid, J. G., Jr., J . Research Natl. Bur. Standards, 38, 137 (1947).
Clegg, D. L., ASAL. CHEY.,22, 48 (1950). Durrum, E. L., J . Am. Chem. Soc., 73, 4875 (1951). Durrum, E. L., J . Colloid Sci., 6, 274 (1951). Durrum, E. L., Science, 113, 66 (1951). Grassmann, W., Naturwissenscha~ten,38, 200 (1951). Haugaard, G., and Kroner, T. D., J . Am. Chem. Soc., 70, 2135 (1948); E. S.Patent 2,555,487 (June 5, 1951). Kirk, P. L., and Duggan, E. L., ASAL. CHEM.,2 4 , 124 (1952). Kraus, K., and Moore, G. E., J . A m . Cheni. Soc., 73, 13 (1951). Kunkel, H. G., and Tiselius, A,; J . Gen. Phusiol., 35, 89 (1951). Lederer, M., Research, 4 , 371 (1951). Lederer, hi., and Ward, F. L., Australian J . Sei., 1 3 , 1 1 4 (1951). McDonald, H. J., J . Chem. Educ., 29, 428 (1952). McDonald, H. J., Urbin, RI. C., and Williamson, 51. B., J . Colloid Sci., 6 , 236 (1951).
Ibid., 2 3 , 25 (1951). Ibid., 24, 356 (1952). Strain, H. H., J . Am. Chtm. Soc., 61, 1292 (1939). Strain, H. H., and Murphy, G. JT., L. CHEM.,24, 50 11952). Strain, H. H., Sato, T. R., and Xorris, W. P., Ibid., 24, 423 (1952).
Strain, H. H., and Sullivan, J. C., Ibid., 23, 810 (1951). Svensson, H., and Brattsten, I., Arlzia Kemi Mineral. G‘col., 1, 401 (1949).
Tiselius, il., hTaturwissenschaften, 37, 25 (1950). Tiselius, A., Nova Acta Reg. SOC.Sci. Upsaliensis, 7 , 27 (1930). Tiselius, A,, Trans. Faraday SOC.,33, 524 (1937). Weber, R., H e k . Chin%.Acta, 34, 2031 (1951). Westman, A. E. R., Scott, 9.E., and Pedley, J. T., dr.4~. CHEM.,24, 1231 (1952). RECEIVEDf o r rei-ieiv July 17, 1952. Accepted December 8, 1952. An unabridged version of this paper is o n record n i t h t h e U. S. Atoniic Energy Commission, AECU-2080, May 1932; Yuclear Science Abstracts. 6 , 543 f 1952).
Colorimetric Determination of Cyanide and Thiocyanate J. 31. KRUSE’
WITH M. G. MELLON Purdue University, Lafayette, Znd.
A study was made at the request of the Federation of Sewage Works Associations for the purpose of improving the method of determining free and combined cyanide and thiocyanate in industrial wastes. The report includes recommendations for isolating the constituents for measurement and the use of a pyridine-pyrazolone reagent for cyanide and a copper-pyridine reagent for thiocyanate. Presented also are results of studies on factors affecting the color-forming reactions, interferences, and attainable precision of the methods. Tests on sewage containing known amounts of various metals and the desired constituents showed the procedures to have satisfactory workability, sensitivity, and precision for the industrial application intended.
C
YASIDE has long been known for its toxicity to man. Recently, more attention has been focused on the effect of very low concentrations of this and similar ions on aquatic life. It has been reported that as little as 0.1 p.p.m. of cyanide is toxic to fish, and a slightly higher concentration will affect the bacteria which normally decompose sewage (8). For these reasons it is necessary to determine cyanide in this concentration range in all types of effluents and industrial wastes. Although several colorimetric methods claim a sensitivity of 1 p.p.m. (1, 4, 6, 10-12), only three of them can be used to determine less than this concentration. The phenolphthalin method (11) does not have the sensitivity of either the pyridine-benzidine ( 1 ) or pyridine-pyrazolone (4, 12) procedures, although it can I
Present address, E. I. du Pont de Nernours & Co.. Ino., Wilmington, Del.
be used to determine less than 1 p.p.m. The latter two procedures are based on the use of a pyridine-benzidine (1) or pyridine-pyrazolone (4, 12) reagent to react nith cyanogen halide formed by treating the sample with bromine or chloramine-T. A comparison of the two methods shoFed that the pyridine-pyrazolone reagent formed a more stable color with cyanide. This reagent has the added advantage of having less substances interfere. The advantages of the pyridine-benzidine reagent are its stability and easy preparation. The pyridine-pyrazolone procedure was finally chosen because it is slightly more sensitive and permits greater precision. It was found necessary to alter somewhat the details of the method as originally described by Epstein. The main problem, hoIvever, was the treatment of the sample necessary to permit the application of the reagent. To facilitate this use in different situations, three separate procedures were developed-namely, methods for the determination of free cyanide, total cyanide, and cyanide in the presence of thiocyanate. A step-by-step set of directions for these separations and subsequent color development for se-ivage analysis has been published (7‘). The object of this paper is t o summarize the developmental work leading up to these procedures. Certain modifications based on further work are introduced, and studies of the reproducibility of these methods are reported. Thiocyanate, although not PO toxic as cyanide, nevertheless is harmful to aquatic life. It therefore is sometimes desirable to determine thiocyanate in a concentration range of about 1 to 10 p.p.m. At higher concentrations, well-known titrimetric methods can be used. On the other hand, the pyridine-pyrazolone reagent can be used for very low concentrations of thiocyanate. However, for the concentration range described, a method which
