Electroosmosis in Paper Electrochromatography With Electrodes on the Paper SCOlT E. WOOD' and HAROLD H. STRAIN Argonne National Laboratory, Lemont, 111. i h aid to electrochrornatographic investigations, the electroosmotic flow of lactic acid solutions in filter paper has been studied with the electrodes fixed to the paper. The flow has been determined by the movement of small zones of noncharged, nonsorbed indicators, as solutions of formaldehyde, hydrogen peroxide, thioacetamide, allylthiourea, and hydroquinone (all detectable by reduction of silver ions), and radioactive sucrose and dextran. With the electrodes on the paper and without reserve of the electrolytic solution, the electroosmotic flow is very small, and the flow rate decreases with the duration of the electrolysis. This small flow also decreaseswith an increase in the paper wetness, and increases with the paper length. In a gi\en paper, the flow across any line transverse to the electrical field is generally uniform, but it varies with the distance from the eIectrodes. The small electroosmotic flow and the decrease of the rate of flow with continued electrolysis have been correlated with the development of resistive forces. These complex forces apparently result from a small change in the distribution of the solution in the paper and possibly from reactions at the electrodes.
E
LECTROCHROMATOGRAPHIC separations in moist paper are usually performed with the electrodes in reservoirs of the electrolytic solution ( 2 , S , 7 ) or with the electrodes attached directly to the paper (.5-i'). With the former electrode arrangement (g), the electroosmotic flow, determined with zones of nonsorbed, noncharged flow indicators, is usually significant and vnriabl~,depending upon a variety of conditions that are diffiw l t to control ( 2 ) . With the latter electrode arrangement, the electroosmotic flow is very small, as revealed by indicator zones or spots containing tritium (HTO) or high concentrations of acids, calcium, or phosphate ions ( 5 ) . -1 search for the conditions that restrict the electroosmotic flow when the electrodes are placed on the paper has led to further investigation of this migration system. To avoid the possibility that the indicators, themselves, might be undergoing electrirnl migration ( A $ ) , either through the solution or along the surface of the paper, several kinds of flow indicators have been cmployed. These indicators are solutions of formaldehyde, hydrogrn peroxide, thioacetamide, allylthiourea, and hydroquinone (all detectable by reduction of silver ions), and radioactive sucrose mid deatritn. The sorbability of these indicators on the paper has :LIFO been determined by standard chromatographic techniques. For the determination of the electroosmosis in different regions of the paper, the indicator zones have been added in special srr;ingements or patterns. The conditions that have been examined include the wetness of the paper, the duration of the electrolysis, the concentration of the electrolytic solution, and tlic trrntment of the paper. MATERIALS
The paper was Eaton-Dikeman Grade 301, 0.03 inch thick. from the same lot described in the preceding article ( 2 ) . For some experiments, it was washed by percolation with 1M nitric acid and water and dried. f
Present address, Illinois Institute of Technology, Chicago, Ill.
1869
The electrolytic solution was usually 0.1M lactic acid. In a few instances, 0.01M acid was employed ( 2 ) . Solutions (0.lM) of the following easily detectable substances in 0.1JI lactic acid were used a s indicators: formaldehyde, hydrogen peroxide, thioacetamide, allylthiourea, and hydroquinone. Radioactive sucrose and dextran were employed a t a concentration of 3.4%. Kunkel and Tiselius (5)have shown that in barbital buffer alone a t pH 8.8, dextran migrates very slightly to the anode. APPARATUS
Electrical migrations were carried out in the moist wrapped in polyethylene sheet 0.005 inch thick. Electroi;::; platinum wire were clamped a t intervals to the ends of the moist paper by paper clips over the polyethylene sheet. During electrolysis, these wrapped paper sheets with the attached electrodes were placed on a Thermopane window cooled with running water (1). PROCEDURE
Sheets of the filter paper were marked with pencil to indicate the position of the electrodes and the points at which indicator solutions were to be added. These marked sheets were placed in the lactic acid solution for 1 to 30 minutes. After removal from the solution, they were blotted between sheets of dry paper under various weights to adjust the amount of the residual electrolytic solution. The indicator solutions (50 pl.) were added to the paper, which was then covered with the polyethylene, and the electrodes were clamped in position. Voltage across the paper, adjusted to 10 volts per centimeter, was measured a t the electrodes. When indicator solutions were added during electrolysis, holes were cut through the polyethylene, and these were covered with a second plastic sheet which was removed momentarily during addition of the indicators. For location of the indicator zones after electrolysis, the moist paper was sprayed with a mixture of equal volumes of 6 N sodium hydroxide and 0.1M silver nitrate in 8% ammonium hydroxide. Hydrogen peroxide yielded a blue spot, the other reducing substances formed brown to black spots. The spots were outlined with a blunt pencil, and the paper was dried uniformly on a flat surface. The radioactive dextran and sucrose were then located autographically in the dry paper. The center of the zones, determined within =kl mm., served as a basis for measurement of movement from the starting point. The sorbability of the indicators was determined by standard chromatographic procedure in large covered beakers using upward flow in paper strips, 15 by 25 cm. The wash liquid, 0.lM lactic acid, was allowed to rise 20 cm., and the acid boundary was located with pH test paper. I n the case of dextran, the yellow advancing front of the lactic acid solution observed in the unwashed paper (8) flowed around the spots before causing them to move, an effect also observed with other surface active substances such as soaps. Presumably the same flow phenomena occurred in the xvashed paper, but the colorless boundary (8) between the acid zone and the wash liquid was not visible. RESULTS
I n washed paper, the R values of the lactic acid and of the indicators were: lactic acid, 1.00; formaldehyde, 0.99; hydrogen peroxide 1.00; thioacetamide, 0.94; allylthiourea, 0.91; hydroquinone, 0.86; dextran, 1.00; sucrose, 1.00. In unwashed paper, the respective values were: 0.82, 0.98, 1.00, 0.93, 0.92, 0.83, 1.00, and 1.00. These results show that, with the exception of hydroquinone, the indicator substances are not sorbed significantly by the paper. The apparent sorption of lactic acid in the unwashed paper may have been due to neutralization of the acid near the leading boundary by cations contained in the paper. The movement of various indicators placed in rows transverse
ANALYTICAL CHEMISTRY
1870
trode. After various periods of electrolysis, hydrogen peroxide was added to three locaPositive movement of zones was toward cathode, negative movement toward anode. Movement values from tions in successive rows beginleft t o right refer, respectively, to formaldehyde, 'hydrogen peroxide, ihioaoetamide, allylthiourea. and hydroquinone ning a t the top of the paper. Paper Lactic Acid The locations for these spots Concn., Per g. paper, Current, Time, H ~ . Movement, M m . No. Size, em. Treatment M g. Ma. were in columns 10 cm. from 22.9 X 30.5 None 0.1 2.64 3.0 19 0, 1. 1, 0, 2 each electrode and across the 22.9 X 3 0 . 5 None 0.1 0.86 3.0 4 3 3 3 3 -3 22.9 X 30.5 Washed 0.1 2.29 3 . 5 -- 2 ; -1: -3: 18 center of the paper sheet as 22.9 X 30.5 0.1 Washed 1.21 6 3 . 0 7 , 10. 10, 7, 6 shown in the figure. The cur20.3 X 30.5 None 0.01 2.40 3 3 . 0 2 , 3, 4, 0. - 1 20.3 X 30.5 None 1.41 0.1 3.1 5 9, 7a, 7 b 8, 9, rent ranged from 25 to 27 ma. Dextran substituted for allylthiourea. The movement and the approxb Sucrose substituted for hydroquinone. imate shape of all the zones are indicated bv the circles in the figure. The movement of to the electrical field is shown in Table I. I n these experiments, the peroxide zones with respect to the duration of the electrolysis one zone of each indicator was placed in a line midway between is shown in Table 111. The movement of the zones of the five the two electrodes (28 cm. apart). The experimental conditions, different indicators was -2, 0, 0, - 2 , and +2 mm. for those including the treatment of the paper, are also summarized in close to the anode and 0, 2, -4, -6, and 0 mm. for those close the table. to the cathode. The movement of the indicators, placed symmetrically in The effect of electrolysis upon the distribution of lactic acid different regions of the paper, was also investigated. I n two solution in paper was determined with two unwashed paper typical examples, paper, 20.3 by 72 cm. (electrodes 70 em. sheets. These papers were 5 cm. by 72 em. and were marked apart), was moistened with lactic acid and blotted, the one paper into sections 5 cm. long. Each paper was impregnated with being dried much more than the other. I n these two papers, lactic acid, blotted gently and then blotted between two strips each indicator was added as a ro\l- of thirteen zones, each zone of similar paper under pressure for 80 minutes. One paper ma8 separated by 5 em. The five parallel rows of the different zones then enclosed in polyethylene while the other was submitted to were separated by 3.8 cm. The movement Iesulting from the electrolysis for 170 minutes; current about 3 ma. Both papers electrolysis is mmmarized in Table 11. In paper 1, the current were cut into 5-em. sections which were rapidly weighed, dried was 15 ma., for 3 hours. I n paper 2, with the least solution, the in air, and reweighed. The average gram of water per section current was 6 ma., for 2.25 hours. of the strip not electrolyzed was 0.96 gram with maximum deviaThe electroosmotic flow and the effect of migrating cations tions of -0.04 and +0.06 gram, Tvhile that of the electrolyzed upon the indicator zones were tested in a piece of untreated paper containing long narrow zones of hydrogen peroxide parallel to the electrodes. For this purpose, the zones of the peroxide were placed 5 em. apart across a paper sheet 21 by 72 em. with electrodes 70 cm. apart. T o form these zones, thirteen in all, a narrow strip of filter paper was moistened with the indicator solution, and this strip was touched across the paper sheet, already moistened with the lactic acid solution. I n addition, separate zones (50 pl.) of cupric, nickel, and silver nitrates (0.1M) were placed in the long zone of hydrogen peroxide nearest the anode. The voltage was applied for 2 hours. The current was 8 ma. The long zones of hydrogen peroxide moved very little and remained straight. Their average movement, from the 70 em * anode toward the cathode in millimeters, was: 0, 1, 2, 3, 7, 11, 'igure 1. Array and Jlovernent of Zones of Electro12, 13, 15, 12, 10, 6, 3. Located with sulfide, the silver ions osmosis Indicators Added during Electroly-sis had passed through three zones of the hydrogen peroxide and +. Original position of spots entered the fourth; the nickel had entered the third; and the 0. Approximate final position and form Zones a t left and right contained five different indicators. cupric ions had partially passed through the second. There was Zones at center and a t each side contained hydrogen peroxide no distortion of the peroxide zones where the zones of the cations added during electrolysis had passed through them. The movement of zones of hydrogen p e r o x i d e a d d e d Table 11. Movement of Zones of Various Indicators during Electrolysis during the electrolysis ivas added in i ~ d Positive nlorement is toward the cathode; negative nlovelnent tol,,ard the anode, ~ determined in a sheet of unrows 5-cm. apart Paper 1 treated paper 35.6 by 72 cm. Movement after Indicator - 3 Hours, 3Irn. After this paper had been 0 -2 -1 0 +2 +3 +2 +4 HzCO moistened with lactic acid and Hz02 +4 +3 +4 CHsCSNHz 0 -1 +2 +l +2 +3 4-3 +4 +3 +3 +? +4 blotted, it was allowed to CaHsNHCSNHz -2 -3 -1 0 +1 +2 +3 +3 +3 +3 stand overnight covered with HOCsHlOH Av. 0 -1 0 +1 +2 +3 +3 +3 +4 +3 +3 +3 +3 polyethylene. The locations Paper 2 for the addition of the zones are Indicator lfovement after 2.25 Hours, 31m, shown as crosses in Figure 1. f 3 +7 +6 +6 +6 +7 +8 +4 +I? HzCO f 3 +1 .4t the beginning of the elecHzOz +3 +1 +4 +6 +8 +8 +g +lo +g +8 +A +4 trolysis, zones of each of the CHGSNH? +4 +1 0 +3 +5 +5 +7 +7 +8 g l +g +j +j C:HsNHCSNH* +3 -1 0 +2 +4 +j +j +7 +7 +8 +5 +4 five silver-reducing indicators HOCEH~OH +3 0 +l +4 +4 +6 +6 +6 +6 +j +7 +6 +4 were placed in two parallel 4v. +3 0 +1 +3 +5 +6 +o +7 +7 +, +a +3 +j columns 5 cm. from each elec-
Table I. Movement of Zones of Various Indicators Placed Midway between Electrodes in Paper Moistened with Lactic Acid
-::
"0'
~
1871
V O L U M E 26, NO, 1 2 , D E C E M B E R 1 9 5 4 strip was 0.99 gram with maximum deviations of -0.13 and $0.12. The smallest quantity of water contained in any section of the tn-o papers was 0.86 gram, and that section was adjacent to the anode. This low value can be due partly to the electrode reaction. In the electrolyzed paper, the five sections nearest the cathode contained about 10% more water than those from the remainder of the strip.
Table 111. Movement of Zones of Hydrogen Peroxide Added during Electrolysis Time of electrolysis is duration of electrolysis after addition of indicator zones. There was no electrolysis before addition of first zone Movement, M m . Electrolysis, dnode Cathode hlin. side Middle side 4 15 300 0 295 285 270
5 4 6
14
13 10
0 0
-1
Flow of the electrolytic solution after electrolysis was tested with zones of hydrogen peroxide added a t the moment the current was discontinued. Several hours thereafter, the position of the zones was determined and found not to have changed more than 1 or 2 mm. However, the addition of 50 pl. of the indicator solution will alter the distribution of the solution already on the paper in the immediate neighborhood of the zone. Electrolysis was without effect upon the size and symmetry of the zones formed by the indicator substances in the moist paper. I n control experiments, the indicator solutions were added to moist sheeh of paper which were wrapped in polyethylene and placed on the cooled Thermopane base. Some were electrolyzed, others were not. Zones formed in all the sheets were the same size, although larger than the initial zones. DISCUSSION
Electroosmosis and Electrochromatography. Under similar migration conditions and within the limits of experimental error, the movement of all the indicator substances is the same (Tables I and 11) Because these substances differ in molecular weight (30 to about 15,000), in chemical composition (Table 11), and in functional groups, their movement is truly indicative of electroosmotic flow, not of electrical migration of the indicators. Because they may be located very easily in the electrical migration systems, and because of their availability and nonsorbability, these flow indicatore may prove useful in many practical applications of electrochromatography. The observations on electroosmotic flow with indicators were substantiated by the determination of the distribution of the solution in the paper. The presence of zones of migrating ions did not cause local electroosmotic effecte as ehown by the passage of various cations through the long zones of hydrogen peroxide. Presumably, the increase in the size of the zones with time is due to diffusion. The observations with the indicators placed across the center of the paper (Table I ) show that the flow increases slightly with a decrease in the quantity of solution per unit weight of the paper. This flow is nearly uniform at all points in any line transverse to the electrical field, but it is not uniform a t other locations in the paper (Table IT). In most papers, the flow is slightly larger near the center of the paper than close to the electrodes, the maximum occurring toward the cathode. The flow is also greater in the longer papers than in the shorter ones (Compare papers 1 and 2 in T a b l a I and IT). There is little difference between the migration in washed and unwashed papers.
The addition of zones of hydrogen peroxide during electrolysis (Table 111) shows that the rate of movement decreases tvith time, presumably approaching a steady state. The symmetry of the circular indicator zones shows that the limited electroosmotic migration cannot be due to electroosmosis in some parts of the capillary system in these zones with equal hydrostatic counterflow in other parts. Similarly, the uniformity of the long zones of hydrogen peroxide evtending across the paper shows that electroosmotic flow does not occur as isolated streams in one region of the paper offset by hydrostatic flow or streams in the opposite direction in other regions. With these possibilities eliminated, the electroosmotic flow must be uniformly opposed and very nearly neutralized by an equal and opposite force. Because there is no reserve or source of the electrolytic solution, a small initial electroosmotic flow must soon establish a difference in the distribution of the electrolytic solution sufficient to oppose the electroosmotic force. Previous work with electrodes on the paper has shown that during electrolysis, significant changes of concentration, pH, and resistance occur near the electrodes ( 5 ) . The acid concentration near the cathode decreases, and the resistance increases. The acid concentration near the anode increases, and the resistance decreases. As the number of faradays used in these experiments is of the order of 10-3,these changes will be small but still effective. They do complicate an explanation of the observed electroosmotic flow as a function of time as well as the slow recovery after the electric current is discontinued. Compared to the experiments with electrode vessels, these blotted papers were significantly drier than those dipping into the electrolytic solution. For example, the blotted paper contained from 0.86 to 2.64 grams of solution per gram of paper (Table I). With the ends of the paper in the solution, however, there were 3 to 3.2 grams of solution per gram of paper for a compression of 0.1 pound per square inch and about 2.8 grams solution per gram of paper for 0.88 pound per square inch ( 2 ) . This migration system, with electrodes fixed on the paper and with weakly dissociated solutes as the electrolytes, serves for many practical electrochromatographic separations. Because of the variation of the wetness of the paper, this system does not provide precise, easily reproducible values for the mobilities of ions. Corrections for the small electroosmotic flow are complicated by the variation of flow in different regions of the paper. Phenomenon of Electroosmosis. From the authors’ earlier investigations, the hydrostatic pressure equivalent to 10 volts per cm. in moist paper is very small ( 2 ) . Moreover, this hydrostatic pressure difference would be created by a very small change in the distribution of the solution in the paper (8). At the moment the voltage is applied, there should be an electroosmotic flow. This movement would increase the quantity of solution in the neighborhood of the cathode and deplete the quantity near the anode, thus producing a nonuniform distribution of the solution in the paper. This nonuniform distribution would create a force opposing that causing the electroosmotic flow and a steady state would be approached. This mechanism, of course, is complicated by the effects of the electrode reactions. These driving and resistive forces and their application to the flow of solution have been examined by means of the thermodynamics of irreversible processes. The resulting equations are
and
where Z is the current; v , the velocity of flow; +, the potential gradient: and F , the opposing forces (hydrostatic and surface). The quantities, L,,, Liz, and Lzz, are the phenomenological coefficients. All the variables and the coefficients are dependent upon the position in the paper and the time. The equations
1872
ANALYTICAL CHEMISTRY
have little practical use, because the values of the variables depend upon the system, and the systems show considerable variations themselves. In addition, no method has been devised by which the opposing forces can be measured directly. ACKNOWLEDGMENT
The radioactive dextran and sucrose, employed as flow indicators, were supplied by Norbert J. Scully of this laboratory. LITERATURE CITED
(1) Chen. Eng. News, 30,4244 (1952). (2) Engelke, J. L., Strain, H. H., and Wood, S. E., ANAL.CHEM.,26, 1864 (1954).
(3) Kunkel, H. G., and Tiselius, A., J . Gen. Physiol., 35, 89 (1951). (4) Longsworth, L. G., J . Am. C h a . SOC.,69, 1288 (1947). (5) Sato, T. R., Kisieleski, W. E., Norris, W. P., and Strain, H. H., ANAL.CHEW,25, 438 (1953). ( 6 ) Strain, H. H., J. Am. Chem. SOC.,61, 1292 (1939). (7) Strain, H. H., Sato, T. R., and Engelke, J. L., ANAL.C H E ~ I26, ., 90 (1954). (8) Wood, S. E., and Strain, H. IT., Ibid.,26, 260 (1954). RECEIVED for review April 7, 1954. Accepted September 17, 1954. Pre. sented in part a t the syinposium on Electromigration in Stabilized Electrolytes, Division of Biological Chemistry and .4merican Association of Clinical Chemists a t the 124th Meeting of the AMERICAN CHEMICAL SOCIETY in Chicago, Ill., September 1953. Scott E Wood nas on leave of absence from Illinoiq Institute of T e r h n o l o ~ y .
Electrical Mobility of Phosphate Ions in Paper Electrochromatography JOHN L. ENGELKE1 and HAROLD H. STRAIN Argonne National Laboratory, Lemont,
111.
The electrical mobility of phosphate ions has been determined in phosphate solutions in paper with zones of radioactive phosphate as the migration indicator. This procedure, which eliminates solution boundaries, shows that the mobility of phosphate varies with the pH. The ions H 9 0 4 - and have nearly equal mobilities, whereas Pod--- migrates almost four times as fast. The electroosmotic flow of the phosphate solutions also varies with the pH. In strongly acid solutions, i t is toward the anode; in weakly acid and alkaline solutions, it is toward the cathode.
I
N WLUTIOSS in paper, M employed in electrochromtttography, there is no simple relationship between the mobilities of ions and their ionic charge. Depending upon the conditions, different ions with the same ionic charge may migrate a t the same or a t different rates. Likewise, the same element in different valence states may also migrate a t different rates (6-8). With many anions, the number of the ionic charges varies with the degree of the dissociation. There is, however, little information concerning the mobility of anions in paper moistened with solutions of different pH values. A succinct report by Sansoni and Klement ( 5 ) shows that the electrical mobility of phosphate ions in paper moistened with organic and inorganic buffers (other than phosphate) varies anomalously with the composition and pH of the solution. In these experimenta, the migration of the phosphate was determined from the movement of small zones of phosphate solutions which were located with molybdate reagent. This observed movement was corrected for the electroosmotic transport determined with separate zones of flow indicators. In this laboratory, the electrical mobility of phosphate ions in moist paper has also been determined aa a function of pH. Here, however, the measurements were made in phosphate solutions with zones of radioactive phosphate as the migration indicator. With this autodetection or self-detection method, there are no solution boundaries in the moist paper, and there is no variation in the kind of buffer (sodium and dissociated phosphoric acid) or in the concentration of total phosphate. With the use of secondary electrodes for the determination of voltage, the electrode reactions are far removed from the paper itself. The observed movement of the phosphate zones was corrected 1 Present address, Department of Chemistry, University of California, Berkeley 4, Calif.
for the electroosmotic transport, which was usually observed with zones of an easily detectable flow indicator-auch as hydrogen peroxide (8). But in the strongly alkaline solutions, owing to the dissociation of the peroxide, hydrazine was used as the flow indicator. In the strongly acid solutions, the electroosmotic flow was toward the anode, whereas in weakly acid and alkaline solutions, it was toward the cathode. In harmony with the earlier investigations (5), the mobility of the phosphate ions was a complex function of their ionic charge and of the p H of the solution. In addition, calculation of the proportions of the several iouic species present a t the different pII values indicates that particular mobilities may be assigned to each species. METHODS
The apparatus utilized for the investigation of electroomiosis in paper ( 2 ) was adapted to the determination of the electrical mobility of the phosphate ions. All the electrolytic solutions were prepared from phosphoric acid or from the sodium salts and their mixtures. All these solutions contained the same concentration (0.01M) of phosphate with variable proportions of sodium. These solutions were used to moisten the paper and to fill the electrode vessels h the same level ( 3 liters each). The pH of the solutions was determined with special calibrated glass electrodes. The temperature of the moist paper was 17' & 2" C. The paper was from a single roll of the Eaton-Dikeman product (Grade 301, 0.03 inch thick). It was different from that em loyed for the study of electroosmosis (2), and it eshibited d d r e n t electroosmotic properties. With this paper, the electroosmotic flow was toward the anode with 0.1M lactic acid as the electrolytic solvent. This paper was cut into strips 4 X 22 inches. These strips were soaked in the phosphate solution and placed between the water-cooled padded plates of the electrolysis cell. Two strips of paper (Eaton-Dikeman, Grade 301, 0.05 inch thick and 2 X 17.5 inches) were moistened and placed along the edges of the cell to help support the upper plate. After 20 minutes, the long paper strip was blotted gently a t the center where the indicators were to be added. The radioactive phosphate, dissolved in the buffer solution (10 pl.), was added to the center of the strip. The flow indicator (0.1M in the buffer solution) was added a s two separate zones (10 pl. each) at each side of the radioactive phosphate. The paper was covered with polyethylene, and voltage (140 f 1 volts, equivalent to 3 volts per cm.) was applied for 3 hours. The ends of the paper Mere then cut off, the cell was opened, and to locate the peroxide zones, the paper was sprayed with a solution of equal parts of sodium hydroxide (6M) and silver nitrate (0.1M) in ammonia (8%). This sprayed paper was dried uniformly on a flat surface, and the radioactive zone was located autographically. Both the phosphate and the peroxide zones were always symmetrical. Conventional chromatographic experiments show that spots
